N-terminal capping modules of ankyrin repeat domains

ABSTRACT

Described herein are proteins comprising an ankyrin repeat domain having an N-terminal capping module with improved properties, as well as corresponding protein libraries, pharmaceutical compositions and nucleic acids encoding such proteins. In other aspects, the disclosure relates to methods using such proteins, corresponding protein libraries or pharmaceutical compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. § 119(a) of EPApplication No. EP20191632.7 filed Aug. 18, 2020, the contents of whichare incorporated herein by reference in their entirety.

SEQUENCE LISTING

The sequence listing of the present application has been submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “2021-04-16-Sequence-Listing-Athebio-AG-190010USPT.txt”,creation date of Apr. 16, 2021, and a size of 70,986 bytes. The sequencelisting submitted via EFS-Web is part of the specification and is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to proteins comprising an ankyrin repeatdomain with an N-terminal capping module conferring improved propertiesto the ankyrin repeat domain, as well as related products and the use ofsuch proteins in methods of treatment.

BACKGROUND OF THE INVENTION

Different classes of specific binding proteins have evolved in nature,the most widely known class being immunoglobulins of vertebrates.Another class of specific binding proteins are repeat proteins. Similarto the role that immunoglobulins play in vertebrates, repeat proteinswere found to be involved in the adaptive immune system of jawless fish.However, repeat proteins play a much wider role beyond this function andmediate protein-protein interactions across all phyla to fulfill diversebiological functions. In fact, they constitute the largest group ofnatural proteins mediating specific binding (e.g. reviewed in Forrer,P., et al., FEBS letters 539, 2-6, 2003). Repeat proteins bind theirtargets via the repeat domain, which is made up of a variable number ofrepeats that stack on each other through their conserved interfaces tocreate the compactly folded repeat domain. Specific target binding isthen achieved through variable residues on the surface of the repeatdomain (Forrer 2003, loc. cit. and WO 2002/020565).

Ankyrin repeat proteins are a well-studied class of repeat proteins. Theankyrin repeat usually comprises 33 amino acid residues forming twoantiparallel alpha-helices and a beta-turn. The folded ankyrin repeatdomain comprising the stacked ankyrin repeats has a right-handedsolenoid structure with a compact hydrophobic core and a large bindingsurface, which allows it to adapt to its respective binding partners(e.g. Binz, H. K., et al., Nat. Biotechnol. 22, 575-582, 2004).

Plückthun and coworkers derived a consensus sequence motif fromnaturally occurring ankyrin repeats (e.g., Binz, H. K., et al., J. Mol.Biol., 332, 489-503, 2003 and WO 2002/020565). The derived ankyrinrepeat consensus motif is 33-amino acid residues long and comprisesfixed and variable positions. The fixed positions correspond mainly toframework residues that are primarily responsible for the structuralintegrity of the ankyrin repeats, in particular, for the interrepeatstacking interactions. The variable positions correspond tosurface-exposed residues that do not strongly contribute to thestructural integrity of the ankyrin repeats; but, are potentiallyinvolved in target binding (though surface-exposed framework residuesmay be involved in target binding too).

Libraries of proteins were then created having an ankyrin repeat domainwith internal ankyrin repeats that were based on such ankyrin repeatconsensus motif (Binz, 2004, loc. cit.). Certain variable positions ofthe consensus motif were randomized in each internal ankyrin repeat toallow for the binding to different targets, thereby creating thediversity of the library. In order to avoid aggregation of ankyrinrepeat domains consisting only of internal ankyrin repeats, the internalankyrin repeats were flanked by an N-terminal capping module and aC-terminal capping module to shield the hydrophobic core of the domainfrom the solvent (Forrer, 2003, loc. cit. and Binz, 2003, loc. cit.).These capping modules were based on the capping repeats of the humanguanine-adenine-binding protein (GA-binding protein). Libraries composedof proteins with ankyrin repeat domains having one, two or threeinternal ankyrin repeats are referred to as an N1C, N2C and N3C library,respectively, wherein the “N” refers to the N-terminal capping module,“C” refers to the C-terminal capping module and the number to the numberof internal ankyrin repeats (Binz, 2003, loc. cit).

Using such a synthetic library of designed ankyrin repeat proteins(DARPins), DARPins against specific targets can be selected with commonselection methods, including phage display, ribosome display and yeastdisplay, and were shown to have very favorable properties. Whiledisplaying binding specificities and affinities that are comparable toimmunoglobulins, DARPins are much more robust and can be easilyengineered into multispecific binding proteins that are easily expressedand purified. DARPins are well studied (e.g. Plückthun, A., Annu. Rev.Pharmacol. Toxicol. 55, 489-511, 2015).

Following the design of the original DARPin library by Plückthun andcoworkers (Binz, 2003, loc. cit. and WO 2002/020565), it was shown thata mutation in the N-terminal capping module can increase thethermostability of an ankyrin repeat domain (WO 2012/069655).

There remains a need to further improve the properties of proteinscomprising an ankyrin repeat domain, such as the thermostability of theankyrin repeat domain.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that specific mutationsin the N-terminal capping module can significantly improve theproperties of an ankyrin repeat domain. In particular, it has been foundthat the amino acid residue present at the position in the N-terminalcapping module, which corresponds to position 15 of SEQ ID NO: 3, is ofkey importance for the thermostability of an ankyrin repeat domain. Ithas also been found that only a few amino acid residues in this positionhave a striking effect on thermostability. Furthermore, the effects ofthese mutations were transferable to ankyrin repeat domains withdiverging sequences, demonstrating the general importance of thisposition in the N-terminal capping module for the thermostability ofankyrin repeat domains.

Accordingly, the present invention provides a protein comprising anankyrin repeat domain, wherein the ankyrin repeat domain comprises anN-terminal capping module with a mutation in the position, whichcorresponds to position 15 of SEQ ID NO: 3.

In a further aspect, the present invention provides a protein librarycomprising such proteins.

In a further aspect, the present invention provides a method ofselection using such protein libraries.

The present invention also provides pharmaceutical compositionscomprising the proteins of the invention, nucleic acids encoding them orvectors or cells comprising said nucleic acids.

In a further aspect, the present invention provides a method ofpreparing a protein of the invention comprising culturing a cell havinga nucleic acid encoding the protein of the invention under conditionsallowing expression thereof and then purifying the expressed protein.

In a further aspect, the present invention relates to the proteins ofthe invention for use in a method of treatment.

Related compositions and methods are also provided, as will be apparentfrom the following detailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Thermal stability of the designed ankyrin repeat proteins P #95and P #101.

Traces from thermal denaturation of proteins P #95 and P #101 are shown.The thermal denaturation is followed by the CD signal at 222 nm in PBSat pH 7.4. The Tm values for P #95 and P #101 were estimated to be 74.5°C. and 85.1° C., respectively.

FF, fraction folded in %; T, temperature in ° C.; P #95 and P #101consist of the amino acid sequences of SEQ ID NO: 95 and SEQ ID NO: 101,respectively. P #101, but not P #95, comprises an improved N-terminalcapping module of the invention.

FIG. 2: Thermal stability of the designed ankyrin repeat proteins P #93and P #94.

Traces from thermal denaturation of P #93 and P #94 are shown. Thethermal denaturation is followed by the CD signal at 222 nm in PBS at pH7.4. The Tm values for P #93 and P #94 were estimated to be 62.1° C. and75.2° C., respectively.

FF, fraction folded in %; T, temperature in ° C.; P #93 and P #94consist of the amino acid sequences of SEQ ID NO:93 and SEQ ID NO:94,respectively. P #94, but not P #93, comprises an improved N-terminalcapping module of the invention.

FIG. 3: Thermal stability of the designed ankyrin repeat proteins P #108and P #109.

Traces from thermal denaturation of proteins P #108 and P #109 areshown. The thermal denaturation is followed by the CD signal at 222 nmin PBS at pH 7.4. The Tm values for P #108 and P #109 were estimated tobe 68.6° C. and 82.8° C., respectively.

FF, fraction folded in %; T, temperature in ° C.; P #108 and P #109consist of the amino acid sequences of SEQ ID NO: 108 and SEQ ID NO:109, respectively. P #109, but not P #108, comprises an improvedN-terminal capping module of the invention.

DETAILED DESCRIPTION OF THE INVENTION

“A”, “an”, and “the” include plural reference unless the context clearlydictates otherwise. Thus, for example, reference to a protein comprisingan ankyrin repeat domain refers to one or more such proteins.

An “ankyrin repeat” refers to a short sequence of amino acid residuesforming a structural motif (e.g., SMART accession number: SM00248).Ankyrin repeats occur in consecutive copies, are involved inprotein-protein interactions and the core of the ankyrin repeat forms ahelix-loop-helix structure.

The term “ankyrin repeat domain” refers to a protein domain comprisingan N-terminal capping module, a C-terminal capping module and one ormore ankyrin repeats in between (also referred to as “internal ankyrinrepeats”). An ankyrin repeat domain has a hydrophobic core, which isshielded on its N- and C-terminal end by N- and C-terminal cappingmodules, respectively, from the solvent. The folded ankyrin repeatdomain has a right-handed solenoid structure with a large bindingsurface that is adaptable to specifically bind targets. The ankyrinrepeat domain is generally very robust and can sustain a significantnumber of mutations, including substitutions, additions and deletions,without destroying its overall structure. The residues that contributeto the structural integrity of the ankyrin repeat domain, including theinterrepeat interactions, are referred to as “framework residues”,whereas the residues that contribute to target binding, either throughdirect interaction with the target or by influencing residues thatdirectly interact with the target, e.g., by stabilizing them, arereferred to as “target interaction residues”. A single amino acidresidue can be both—a framework and a target interaction residue—at thesame time and framework residues and target interaction residues may befound not only in the internal ankyrin repeats, but also the N-terminalcapping module and/or the C-terminal capping module.

The internal ankyrin repeats contribute to the structural stability ofthe ankyrin repeat domain due to their stacking interaction with theneighboring repeats. An internal ankyrin repeat usually consists of 33amino acid residues.

The capping modules have a hydrophobic inside surface that is suitablefor interacting with the adjacent internal ankyrin repeat and ahydrophilic outside surface to shield the hydrophobic core from thesolvent. In some embodiments, the N-terminal capping module and/or theC-terminal capping module are a N-terminal capping repeat and/orC-terminal capping repeat, respectively, which have a similar or thesame fold as the adjacent internal ankyrin repeat(s) and/or sequencesimilarities to said adjacent internal ankyrin repeat(s).

The terms “binding”, “specific binding” or the like when used inreference to a target mean a binding interaction that is measurablydifferent from a non-specific interaction, e.g., the interaction with acontrol molecule that is unrelated to the specific target. Controlmolecules that are commonly used to measure such non-specificinteraction include bovine serum albumin, bovine casein and Escherichiacoli maltose binding protein (unless, of course, the respective proteinswere the target for the specific binding). In certain instances, theterms “binding”, “specific binding” or the like mean that only thetarget is bound and substantially no other molecule. Specific bindingcan be determined, for instance, by measuring the dissociation constant(Kd) for the target and/or by comparing the binding to the target withthe binding to a control molecule. The Kd can be measured by variousconventional techniques, such as isothermal titration calorimetry,radioligand binding assay, fluorescence energy resonance transfer, andsurface plasmon resonance. The binding specificity is generally measuredin standardized solutions, such as PBS. For instance, the Kd for thetarget in PBS is at least 10, at least 10², at least 10³ or at least 10⁴times lower than the corresponding Kd for a control molecule that isunrelated to the specific target.

The term “designed ankyrin repeat protein” or “DARPin” refers to anon-natural protein comprising an ankyrin repeat domain. In someembodiments, such a DARPin has a repeat sequence motif that was derivedfrom natural ankyrin repeats, e.g. by consensus design (see, e.g.,Forrer et al., 2004 Chem Bio Chem, 5, 2, 183-189 and Binz 2003, loc.cit).

The term “fraction of refolded ankyrin repeat domains after thermaldenaturation” refers to the fraction of ankyrin repeat domains thatrefold into their native state after thermal denaturation.

The term “library” as used in reference to a protein or nucleic acidlibrary refers to a collection of proteins and nucleic acids,respectively.

The term “melting temperature” or “Tm” refers to the temperature atwhich 50% of the protein is unfolded in a certain buffer, e.g. PBS.

The term “modification”, as used in reference to a specific amino acidsequence (e.g. the amino acid sequence of an internal ankyrin repeat orcapping module), refers to one or more modification(s) of said aminoacid sequence selected from the group consisting of deletions,insertions and/or substitutions. In some embodiments, the number ofdeletions and insertions is limited, for instance, to a combined numberof deletions and insertions of not more than three, not more than two ornot more than one of the total number of modification(s). Accordingly,if there is a total number of not more than 9 modifications, the numberof deletions and insertions of those not more than 9 modifications maybe limited to a combined number of insertions and deletions of not morethan two. In some embodiments, the modification(s) are substitution(s)only. A substitution can be a substitution of an amino acid residuewith, e.g., any of the naturally occurring amino acid residues. In someembodiments, the substitution of an amino acid residue is with an aminoacid residue selected from the group consisting of A, D, E, F, H, I, K,L, M, N, Q, R, S, V, W and Y. In some embodiments, the substitution ofan amino acid residue is with an amino acid residue selected from thegroup consisting of A, D, E, H, I, K, L, Q, R, S, V and Y. The followingamino acid residues may, for instance, be particularly suitable for therespective position of the N-terminal capping module:

TABLE 1 Position Amino acid residue 1 A, E, N, Q, G, S, T, K, D, R, H, C2 E, L, Q, M, K, R, C 3 G, D, S, A, C 4 A, R, T, S, N, Q, K, D, E, H, C5 A, R, T, S, N, Q, K, D, E, H, C 6 A, L, N, S, D, C 7 L, I, V, A, N, T,S, D, C 8 E, D, Q, A, N, S, T, K, R, H, C 9 A 10 V, S, A, C 11 L, S, Q,K, R, A, H, D, E, C, T, N, F, W, Y 12 K, R, A, T, S, N, Q, D, E, H, C 13G, C 14 N, S, T, A, D, E, K, Q, R, H, C 15 M, I, T, A, L, V, S, N, D, Q,K, R, E, C 16 D, A, N, Q, S, T, K, E, R, H, C 17 D, A, N, Q, S, T, K, E,R, H, C 18 T, A, S, I, L, V, C 19 R, E, D, K, A, N, Q, S, T, H, C 20 N,K, R, T, S, E, Q, A, D, H, C, I, V 21 N, S, L, A, C 22 I, A, V, M, T, L,S, N, C 23 R, S, Q, K, N, A, E, D, H, C 24 A, H, K, R, L, I, V, C, G 25G 26 A 27 N, D, C 28 T, V, S, P, A, C 29 D, N, C 30 A, C

An amino acid substitution may be a conservative or non-conservativesubstitution. In some embodiments, substitutions only relate toconservative amino acid substitutions. A conservative amino acidsubstitution typically involves exchanging an amino acid residue by adifferent one having similar biophysical properties. For instance, theamino acid residues with a positively charged sidechain K, R and H, theamido acids with negatively charged sidechain E and D, the amino acidresidue with a polar side chain T and S, the amino acid residues with anaromatic sidechain F, W or Y or the amino acid residues with a non-polarsidechain A, V, L, I and M may be substituted with one another.

The term “PBS” refers to phosphate-buffered saline containing 137 mMNaCl, 10 mM phosphate and 2.7 mM KCl and having a pH of 7.4.

The term “percent (%) sequence identity” with respect to a specificamino acid sequence (e.g. the amino acid sequence of a N-terminalcapping module of the invention) is defined as the percentage of aminoacid residues in a candidate sequence that is identical with the aminoacid residues in the specific amino acid sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2 or ALIGN.Those skilled in the art can determine appropriate parameters formeasuring alignment, including any algorithms needed to achieve maximalalignment over the full length of the sequences being compared. Furtherexamples of how to determine the percentage of sequence identity can befound in WO 2009/058564 A2, page 93, line 14 to page 102, line 5.

The term “pharmaceutically acceptable carrier” refers to buffers,carriers, and other excipients suitable for use in contact with tissuesof humans and/or animals without excessive toxicity, allergic response,irritation, or other problem or complication, commensurate with areasonable benefit/risk ratio. The carrier(s) should be “acceptable” inthe sense of being compatible with the other ingredients of theformulations and not deleterious to the recipient. Pharmaceuticallyacceptable carriers include buffers, solvents, dispersion media,coatings, isotonic and absorption delaying agents, and the like, thatare compatible with pharmaceutical administration.

The term “pharmaceutical composition” refers to a composition comprisingat least one active agent and at least one pharmaceutically acceptablecarrier. A pharmaceutical composition is generally formulated andadministered to exert a pharmaceutically useful effect while minimizingundesirable side effects.

If the term “position” is used without further reference to a particularamino acid sequence, then it refers to the corresponding amino acidposition of SEQ ID NO: 3. Furthermore, “corresponding” in this contextmeans that the amino acid residue aligns with the indicated position ofa specific sequence in a sequence alignment. Alignment for purposes ofdetermining which amino acid residue corresponds to which position of aspecific sequence can be achieved in various ways that are within theskill in the art, for instance, using publicly available computersoftware such as BLAST, BLAST-2 or ALIGN. Those skilled in the art candetermine appropriate parameters for measuring alignment, including anyalgorithms needed to achieve maximal alignment over the full length ofthe sequences being compared. Further examples of how to align twosequences can be found in WO 2009/058564 A2, page 94, line 7 to page 96,line 28.

The term “recombinant”, as used in reference to a protein, refers to aprotein produced from a recombinant nucleic acid. A “recombinant nucleicacid” refers to nucleic acid molecules formed by laboratory methods ofgenetic recombination or gene synthesis.

The term “substantially identical”, as used in reference to a specificamino acid sequence (e.g. the N-terminal capping module of the inventionor an internal ankyrin repeat), refers to amino acid sequences having(1) at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or 100% sequence identity to the specific amino acidsequence or (2) up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, upto 3, up to 2, up to 1 or no modifications, as compared to the specificamino acid sequence.

The term “target”, as used, for instance, in conjunction with thespecific binding property of an ankyrin repeat domain comprised in aprotein, refers to any substance or structure. It may refer to a singlemolecule, such as a protein, small-molecule or sugar, as well ascomplexed molecules, such as interacting proteins or proteins binding tonon-proteinaceous compounds. It may also refer to more macromolecularstructures, such as cells, tissues, viruses or bacteria.

The terms “treating” or “treatment” of a disease, condition or symptomrefers to obtaining therapeutic and/or prophylactic benefit, includingalleviating, ablating, ameliorating, or preventing a disease, conditionor symptoms, preventing additional symptoms, ameliorating or preventingthe underlying metabolic causes of symptoms, inhibiting the disease orcondition, e.g., arresting or slowing down the development of thedisease or condition, relieving the disease or condition, causingregression of the disease or condition, relieving a condition caused bythe disease or condition, or stopping the symptoms of the disease orcondition.

Naturally occurring ankyrin repeat domains normally have capping modulesto shield their hydrophobic cores from the solvent. In line with this,earlier developed recombinant proteins having ankyrin repeat domainslikewise comprised capping modules with such a shielding function at theN- and C-terminal ends of the ankyrin repeat domain (e.g. Binz, 2003,loc. cit. and Binz, 2004, loc. cit.). The capping modules that werefirst used were derived from the murine GABPβ1, which is a naturallyoccurring ankyrin repeat protein (PDB: 1AWC_B). Binz et al. 2003 (loc.cit.) already introduced some modifications to the naturally occurringcapping modules in order to adapt the capping modules for binding to theinternal ankyrin repeats and for cloning purposes. The N-terminalcapping module of Binz et al. 2003 (loc. cit.) is reflected by SEQ IDNO: 3 and the C-terminal capping module of Binz et al. 2003 (loc. cit.)is reflected by SEQ ID NO: 83.

WO 2012/069655 relates to further modified N-terminal and C-terminalcapping modules of the ankyrin repeat domain. The N-terminal cappingmodules disclosed in WO 2012/069655 include the two N-terminal cappingmodules reflected by SEQ ID NO: 1 and SEQ ID NO: 2 (corresponding to SEQID NO: 15 and 14 of WO 2012/069655, respectively, without the twooptionally missing N-terminal amino acid residues). Another exemplaryN-terminal capping module disclosed in the prior art includes the onereflected by SEQ ID NO: 4 corresponding to an amino acid sequencecomprised in the N-terminal capping module of SEQ ID NO: 56 of WO2016/156596 A1.

Using in silico structure analysis, the present inventors determinedthose amino acid residues that would appear to be most suitable for eachposition of the N-terminal capping module of the ankyrin repeat domain.In particular, the various amino acid residues shown in Table 1 wereconsidered to be particularly suitable based on the in silico analysis.

Based on the structural analysis, amino acid residues in variouspositions were tested by in vitro experimentation. Among the many testedmutations of the N-terminal capping module, there was one position whichwas surprisingly found to be particularly important for thethermostability of the ankyrin repeat domain—the position correspondingto position 15 of SEQ ID NO: 3 (also simply referred to as position 15herein without referencing SEQ ID NO: 3).

Accordingly, the protein of the invention comprises an ankyrin repeatdomain that has an N-terminal capping module with a mutation in position15.

In some embodiments, the ankyrin repeat domain of the protein of theinvention has improved properties, which may include improvedthermostability, improved storage stability, improved thermodynamicstability (defined as the difference in free energy between the foldedand unfolded states), improved folding and/or refolding properties (suchas a higher fraction of refolded ankyrin repeat domains after thermaldenaturation), reduced aggregation propensity and lower in vivoimmunogenicity risk.

In some embodiments, the N-terminal capping module has an amino acidresidue at position 15 selected from the group consisting of I, T, A, V,L, M, S, Q and R. In some embodiments, the N-terminal capping module hasan amino acid residue at position 15 selected from the group consistingof I, T, A, V, L, M and S. In some embodiments, the N-terminal cappingmodule has an amino acid residue at position 15 selected from the groupconsisting of I, T, A, V, L and M. In some embodiments, the N-terminalcapping module has I at position 15. In some embodiments, the N-terminalcapping module has T at position 15. In some embodiments, the N-terminalcapping module has A at position 15. In some embodiments, the N-terminalcapping module has V at position 15. In some embodiments, the N-terminalcapping module has L at position 15. In some embodiments, the N-terminalcapping module has M at position 15.

In some embodiments, the N-terminal capping module has, apart from themutation in position 15, an amino acid residue of Table 1 in one or moreof the other positions.

In some embodiments, the N-terminal capping module further has an aminoacid residue at position 3 selected from the group consisting of G, A, Dand S. In some embodiments, the N-terminal capping module has an aminoacid residue at position 3 selected from A and S. In some embodiments,the N-terminal capping module has A at position 3. In some embodiments,the N-terminal capping module has S at position 3.

In some embodiments, the N-terminal capping module further has an aminoacid residue at position 8 selected from the group consisting of E, D,Q, N, S, T, K and R. In some embodiments, the N-terminal capping modulehas Q at position 8.

In some embodiments, the N-terminal capping module further has an aminoacid residue at position 11 selected from the group consisting of L, S,Q, K, R, A, H, D and E. In some embodiments, the N-terminal cappingmodule has an amino acid residue at position 11 selected from K, E, Q, Aand L. In some embodiments, the N-terminal capping module has A atposition 11.

In some embodiments, the N-terminal capping module further has an aminoacid residue at position 16 selected from the group consisting of D, A,N, Q, G, S, T, K, E and R. In some embodiments, the N-terminal cappingmodule has E at position 16.

In some embodiments, the N-terminal capping module further has an aminoacid residue at position 17 selected from the group consisting of D, A,N, Q, G, S, T, K, E and R. In some embodiments, the N-terminal cappingmodule has A at position 17.

In some embodiments, the N-terminal capping module further has an aminoacid residue at position 19 selected from the group consisting of R, E,D, K, A, N, Q, S, T and H. In some embodiments, the N-terminal cappingmodule has E at position 19.

In some embodiments, the N-terminal capping module further has an aminoacid residue at position 20 selected from the group consisting of I, V,N, K, R, T, S, E, Q, A, D and H. In some embodiments, the N-terminalcapping module has Q at position 20.

In some embodiments, the N-terminal capping module further has an aminoacid residue at position 22 selected from the group consisting of L, V,I and A. In some embodiments, the N-terminal capping module further hasan amino acid residue at position 22 selected from the group consistingof L, V and I. In some embodiments, the N-terminal capping module has Lat position 22. In some embodiments, the N-terminal capping module has Vat position 22. In some embodiments, the N-terminal capping module has Iat position 22. In some embodiments, the N-terminal capping module has Aat position 22.

In some embodiments, the N-terminal capping module has L at position 15and I at position 22. In some embodiments, the N-terminal capping modulehas M at position 15 and I at position 22. In some embodiments, theN-terminal capping module has T at position 15 and I at position 22. Insome embodiments, the N-terminal capping module has I at position 15 andI at position 22. In some embodiments, the N-terminal capping module hasA at position 15 and I at position 22. In some embodiments, theN-terminal capping module has V at position 15 and I at position 22.

In some embodiments, the N-terminal capping module has L at position 15and L at position 22. In some embodiments, the N-terminal capping modulehas M at position 15 and L at position 22. In some embodiments, theN-terminal capping module has T at position 15 and L at position 22. Insome embodiments, the N-terminal capping module has I at position 15 andL at position 22. In some embodiments, the N-terminal capping module hasA at position 15 and L at position 22. In some embodiments, theN-terminal capping module has V at position 15 and L at position 22.

In some embodiments, the N-terminal capping module has L at position 15and V at position 22. In some embodiments, the N-terminal capping modulehas M at position 15 and V at position 22. In some embodiments, theN-terminal capping module has T at position 15 and V at position 22. Insome embodiments, the N-terminal capping module has I at position 15 andV at position 22. In some embodiments, the N-terminal capping module hasA at position 15 and V at position 22. In some embodiments, theN-terminal capping module has V at position 15 and V at position 22.

In some embodiments, the ankyrin repeat domain of the protein of theinvention has an improved thermostability, such as a higher meltingtemperature and/or a higher fraction of refolded ankyrin repeat domainsafter thermal denaturation, as compared to a reference ankyrin repeatdomain having the same amino acid sequence except for the mutation inposition 15 of the N-terminal capping module.

Methods for measuring the thermostability of a protein or a proteindomain are well-known to the person skilled in the art. For instance,the thermostability can be measured by a thermal shift assay,differential scanning calorimetry and circular dichroism (CD). Anotherpossible approach is to use differential scanning fluorimetry (e.g.Nielsen et al., 2007, Nat Protoc. 2, 9:2212-21). In this method,unfolding of the protein is measured with a fluorescent dye that bindsto hydrophobic parts of the protein. As the protein unfolds, morehydrophobic parts become exposed causing an increase in fluorescence andvice versa.

This method therefore allows to conveniently monitor the refoldingproperties of a protein and to determine its melting temperature, whichcorresponds to the midpoint of the fluorescence transition curve. Therefolding properties and melting temperature of a protein can also bemeasured by CD spectroscopy, whereby the thermal melting curve of theprotein is determined by measuring the CD signal at 222 nm. For purposesof measuring the thermostability, the protein may be dissolved in PBS.For example, the thermostability of a helical protein can be determinedby measuring the CD signal of the protein at 222 nm while slowly heatingthe protein at a concentration of 0.01 mM in PBS pH 7.4 from 20° C. to95° C. using a temperature ramp of 1° C. per min.

In some embodiments, the increase in melting temperature of the ankyrinrepeat domain of the invention is at least 1° C., at least 2° C., atleast 3° C., at least 4° C. or at least 5° C., as compared to thereference ankyrin repeat domain.

In some embodiments, the fraction of the refolded ankyrin repeat domainsafter thermal denaturation is at least 1%, at least 5%, at least 10% orat least 20% higher, as compared to the reference ankyrin repeat domain.

In some embodiments, the ankyrin repeat domain has a higher meltingtemperature and/or higher fraction of refolded ankyrin repeat domainsafter thermal denaturation. In some embodiments, the ankyrin repeatdomain has a higher melting temperature and/or higher fraction ofrefolded ankyrin repeat domains after thermal denaturation than areference ankyrin repeat domain with the same amino acid sequence exceptfor position 15 of the N-terminal capping module that is a differentamino acid residue in the reference ankyrin repeat domain compared tothe ankyrin repeat domain of the protein of the invention. In someembodiments, the amino acid residue in position 15 of the referenceankyrin repeat domain is selected from the group consisting of E, D, G,H, K and N. In some embodiments, the amino acid residue in position 15of the reference ankyrin repeat domain is E. In some embodiments, theamino acid residue in position 15 of the reference ankyrin repeat domainis D.

Unless specified, the sequence of the ankyrin repeat domain is notparticularly limited. In particular, the ankyrin repeat domain allowsfor a large sequence variation while preserving the overall structureand function of the domain.

In some embodiments, the N-terminal capping module comprises an aminoacid sequence that is substantially identical to an amino acid sequenceselected from the group consisting of SEQ ID NOs: 1 to 76 and 110. Insome embodiments, the N-terminal capping module comprises an amino acidsequence that is substantially identical to an amino acid sequenceselected from the group consisting of SEQ ID NOs: 7 to 16. In someembodiments, the N-terminal capping module comprises an amino acidsequence that is substantially identical to an amino acid sequenceselected from the group consisting of SEQ ID NOs: 17 to 26. In someembodiments, the N-terminal capping module comprises an amino acidsequence that is substantially identical to an amino acid sequenceselected from the group consisting of SEQ ID NOs: 27 to 36. In someembodiments, the N-terminal capping module comprises an amino acidsequence that is substantially identical to an amino acid sequenceselected from the group consisting of SEQ ID NOs: 37 to 46. In someembodiments, the N-terminal capping module comprises an amino acidsequence that is substantially identical to an amino acid sequenceselected from the group consisting of SEQ ID NOs: 47 to 56. In someembodiments, the N-terminal capping module comprises an amino acidsequence that is substantially identical to an amino acid sequenceselected from the group consisting of SEQ ID NOs: 57 to 66 and 110. Insome embodiments, the N-terminal capping module comprises an amino acidsequence that is substantially identical to an amino acid sequenceselected from the group consisting of SEQ ID NOs: 67 to 76.

In some embodiments, the N-terminal capping module comprises an aminoacid sequence that has at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or 100% sequence identity to anamino acid sequence selected from the group consisting of SEQ ID NOs: 1to 76 and 110. In some embodiments, the N-terminal capping modulecomprises an amino acid sequence that has at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or 100% sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 7 to 16. In some embodiments, the N-terminal capping modulecomprises an amino acid sequence that has at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or 100% sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 17 to 26. In some embodiments, the N-terminal capping modulecomprises an amino acid sequence that has at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or 100% sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 27 to 36. In some embodiments, the N-terminal capping modulecomprises an amino acid sequence that has at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or 100% sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 37 to 46. In some embodiments, the N-terminal capping modulecomprises an amino acid sequence that has at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or 100% sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 47 to 56. In some embodiments, the N-terminal capping modulecomprises an amino acid sequence that has at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or 100% sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 57 to 66 and 110. In some embodiments, the N-terminalcapping module comprises an amino acid sequence that has at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or100% sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 67 to 76.

In some embodiments, the N-terminal capping module comprises an aminoacid sequence that is identical to an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 7 to 76 and 110. In someembodiments, the N-terminal capping module comprises an amino acidsequence that is identical to an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 7 to 16. In some embodiments, theN-terminal capping module comprises an amino acid sequence that isidentical to an amino acid sequence selected from the group consistingof SEQ ID NOs: 17 to 26. In some embodiments, the N-terminal cappingmodule comprises an amino acid sequence that is identical to an aminoacid sequence selected from the group consisting of SEQ ID NOs: 27 to36. In some embodiments, the N-terminal capping module comprises anamino acid sequence that is identical to an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 37 to 46. In some embodiments,the N-terminal capping module comprises an amino acid sequence that isidentical to an amino acid sequence selected from the group consistingof SEQ ID NOs: 47 to 56. In some embodiments, the N-terminal cappingmodule comprises an amino acid sequence that is identical to an aminoacid sequence selected from the group consisting of SEQ ID NOs: 57 to 66and 110. In some embodiments, the N-terminal capping module comprises anamino acid sequence that is identical to an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 67 to 76. In some embodiments,the N-terminal capping module comprises an amino acid sequence that isidentical to SEQ ID NO: 110.

In some embodiments, the N-terminal capping module comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs: 7 to 16or from a variant of any one of SEQ ID NOs: 7 to 16 with up to 9, up to8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1modifications outside position 15, and wherein X at position 15 of SEQID NOs: 7 to 16 or said variants thereof is an amino acid residue asdefined above for position 15. In some embodiments, the variants of SEQID NOs: 7 to 16 have up to 9 modifications. In some embodiments, thevariants of SEQ ID NOs: 7 to 16 have up to 8 modifications. In someembodiments, the variants of SEQ ID NOs: 7 to 16 have up to 7modifications. In some embodiments, the variants of SEQ ID NOs: 7 to 16have up to 6 modifications. In some embodiments, the variants of SEQ IDNOs: 7 to 16 have up to 5 modifications. In some embodiments, thevariants of SEQ ID NOs: 7 to 16 have up to 4 modifications. In someembodiments, the variants of SEQ ID NOs: 7 to 16 have up to 3modifications. In some embodiments, the variants of SEQ ID NOs: 7 to 16have up to 2 modifications. In some embodiments, the variants of SEQ IDNOs: 7 to 16 have up to 1 modification. In some embodiments, there areno variants of SEQ ID NOs: 7 to 16. In some embodiments, themodifications of SEQ ID NOs: 7 to 16 do not include more than a combinednumber of deletions and insertions of 3. In some embodiments, themodifications of SEQ ID NOs: 7 to 16 do not include more than a combinednumber of deletions and insertions of 2. In some embodiments, themodifications of SEQ ID NOs: 7 to 16 do not include more than a combinednumber of deletions and insertions of 1. In some embodiments, themodifications of SEQ ID NOs: 7 to 16 are only substitutions. In someembodiments, the variants of SEQ ID NOs: 7 to 16 comprise one or moresubstitutions with an amino acid residue of Table 1.

In some embodiments, the N-terminal capping module comprises an aminoacid sequence with amino acid residues as indicated for the positions 1to 30 in Table 1. For instance, the amino acid residue at position 1 isselected from the group consisting of A, E, N, Q, G, S, T, K, D, R andH, and so on. In some embodiments, the N-terminal capping modulecomprises an amino acid sequence with amino acid residues as indicatedfor the positions 1 to 30 in Table 1 except for position 15 that isselected from the group consisting of I, T, A, V, L and M. In someembodiments, the N-terminal capping module comprises an amino acidsequence with amino acid residues as indicated for the positions 1 to 30in Table 1 except for position 15 that is selected from the groupconsisting of I, T, A, V, L and M or a variant thereof with up to 9, upto 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1modifications outside position 15.

The N-terminal capping module may further comprise a sequence N-terminalto the amino acid sequences defined in SEQ ID NOs: 1 to 76 and 110 (orthe sequence variants thereof defined herein). For instance, suchsequence could be a dipeptide comprising amino acid residues selectedfrom the group consisting of D, A, E, N, Q, S, T, K, R and H, such asthe dipeptide GS, DA, EA, AA, AD, AE, NA, AN, PT, TP, AT or TA. In someembodiments, the dipeptide is directly N-terminal to the amino acidsequences defined in SEQ ID NOs: 1 to 76 and 110 (or the sequencevariants thereof defined herein). Such dipeptide sequence primarilyserves as a linker to connect the ankyrin repeat domain to the furtherpeptide sequence of the protein or as an extended alpha-helix of theN-terminal capping module.

It is understood that for those embodiments of the N-terminal cappingmodule, which are defined by a certain amino acid residue(s) in, e.g.,position 15, as well as a minimal sequence identity to an amino acidsequence or a defined number of modifications compared to an amino acidsequence, both conditions need to be fulfilled. For instance, anN-terminal capping module having I in position 15 and at least 70%sequence identity to SEQ ID NOs: 7 to 16, only relates to suchembodiments wherein the N-terminal capping module has I in position 15and, at the same time, at least 70% sequence identity to one or more ofSEQ ID NOs: 7 to 16.

In some embodiments, the internal ankyrin repeats of the ankyrin repeatdomain consist of 33 amino acid residues.

In some embodiments, the internal ankyrin repeat(s) of the ankyrinrepeat domain of the invention comprise an amino acid sequence that issubstantially identical to an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 77 to 82 or from the group consisting ofSEQ ID NOs: 78 to 82. In some embodiments, the internal ankyrinrepeat(s) comprise an amino acid sequence that has at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or100% sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 77 to 82 or from the group consisting of SEQID NOs: 78 to 82. In some embodiments, the internal ankyrin repeat(s)comprise an amino acid sequence selected from the group consisting ofSEQ ID NOs: 77 to 82 or from a variant of any one of SEQ ID NOs: 77 to82 with up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3,up to 2 or up to 1 modifications. In some embodiments, the internalankyrin repeat(s) comprise an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 78 to 82 or from a variant of any one ofSEQ ID NOs: 78 to 82 with up to 9, up to 8, up to 7, up to 6, up to 5,up to 4, up to 3, up to 2 or up to 1 modifications.

In some embodiments, the internal ankyrin repeat adjacent to theN-terminal capping module of the ankyrin repeat domain of the inventioncomprises an amino acid sequence that is substantially identical to anamino acid sequence selected from the group consisting of SEQ ID NOs: 77to 82 or from the group consisting of SEQ ID NOs: 78 to 82. In someembodiments, the internal ankyrin repeat adjacent to the N-terminalcapping module comprises an amino acid sequence that has at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or100% sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 77 to 82 or from the group consisting of SEQID NOs: 78 to 82. In some embodiments, the internal ankyrin repeatadjacent to the N-terminal capping module comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 77 to 82 orfrom a variant of any one of SEQ ID NOs: 77 to 82 with up to 9, up to 8,up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1modifications. In some embodiments, the internal ankyrin repeat adjacentto the N-terminal capping module comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 78 to 82 or from avariant of any one of SEQ ID NOs: 78 to 82 with up to 9, up to 8, up to7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 modifications.

In some embodiments, the internal ankyrin repeats of an ankyrin repeatdomain of the invention share a high degree of sequence identity orsequence similarity. In some embodiments, the internal ankyrin repeat(s)share at least 70%, at least 75%, at least 80%, at least 85%, at least90% or at least 95% sequence identity.

In some embodiments, the C-terminal capping module of the ankyrin repeatdomain of the invention comprises an amino acid sequence that issubstantially identical to an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 83 to 92, 111 and 112. In someembodiments, the C-terminal capping module comprises an amino acidsequence that has at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, or 100% sequence identity to an aminoacid sequence selected from the group consisting of SEQ ID NOs: 83 to92, 111 and 112. In some embodiments, the C-terminal capping modulecomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 83 to 92, 111 and 112 or from a variant of any one of SEQ IDNOs: 83 to 92, 111 and 112 with up to 9, up to 8, up to 7, up to 6, upto 5, up to 4, up to 3, up to 2 or up to 1 modifications.

In some embodiments, the N-terminal capping module of the ankyrin repeatdomain of the invention comprises an amino acid sequence that issubstantially identical to an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 7 to 16 and the internal ankyrinrepeat(s) comprise an amino acid sequence that is substantiallyidentical to an amino acid sequence selected from the group consistingof SEQ ID NOs: 77 to 82. In some embodiments, the N-terminal cappingmodule comprises an amino acid sequence that has at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 7 to 16 and the internal ankyrin repeat(s)comprise an amino acid sequence that has at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or 100% sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 77 to 82. In some embodiments, the N-terminal capping modulecomprises an amino acid sequence that has at least 70% sequence identityto an amino acid sequence selected from the group consisting of SEQ IDNOs: 7 to 16 and the internal ankyrin repeat(s) comprise an amino acidsequence that has at least 70% sequence identity to an amino acidsequence selected from the group consisting of SEQ ID NOs: 77 to 82. Insome embodiments, the N-terminal capping module comprises an amino acidsequence that has at least 75% sequence identity to an amino acidsequence selected from the group consisting of SEQ ID NOs: 7 to 16 andthe internal ankyrin repeat(s) comprise an amino acid sequence that hasat least 75% sequence identity to an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 77 to 82. In some embodiments, theN-terminal capping module comprises an amino acid sequence that has atleast 80% sequence identity to an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 7 to 16 and the internal ankyrinrepeat(s) comprise an amino acid sequence that has at least 80% sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 77 to 82. In some embodiments, the N-terminal capping modulecomprises an amino acid sequence that has at least 85% sequence identityto an amino acid sequence selected from the group consisting of SEQ IDNOs: 7 to 16 and the internal ankyrin repeat(s) comprise an amino acidsequence that has at least 85% sequence identity to an amino acidsequence selected from the group consisting of SEQ ID NOs: 77 to 82. Insome embodiments, the N-terminal capping module comprises an amino acidsequence that has at least 90% sequence identity to an amino acidsequence selected from the group consisting of SEQ ID NOs: 7 to 16 andthe internal ankyrin repeat(s) comprise an amino acid sequence that hasat least 90% sequence identity to an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 77 to 82. In some embodiments, theN-terminal capping module comprises an amino acid sequence that has atleast 95% sequence identity to an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 7 to 16 and the internal ankyrinrepeat(s) comprise an amino acid sequence that has at least 95% sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 77 to 82. In some embodiments, the N-terminal capping modulecomprises an amino acid sequence that is identical to an amino acidsequence selected from the group consisting of SEQ ID NOs: 7 to 16 andthe internal ankyrin repeat(s) comprise an amino acid sequence that isidentical to an amino acid sequence selected from the group consistingof SEQ ID NOs: 77 to 82.

In some embodiments, the N-terminal capping module comprises an aminoacid sequence that is substantially identical to an amino acid sequenceselected from the group consisting of SEQ ID NOs: 7 to 16, the internalankyrin repeat(s) comprise an amino acid sequence that is substantiallyidentical to an amino acid sequence selected from the group consistingof SEQ ID NOs: 77 to 82, and the C-terminal capping module comprises anamino acid sequence that is substantially identical to an amino acidsequence selected from the group consisting of SEQ ID NOs: 83 to 92, 111and 112. In some embodiments, the N-terminal capping module comprises anamino acid sequence that has at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 95%, or 100% sequence identity toan amino acid sequence selected from the group consisting of SEQ ID NOs:7 to 16, the internal ankyrin repeat(s) comprise an amino acid sequencethat has at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or 100% sequence identity to an amino acidsequence selected from the group consisting of SEQ ID NOs: 77 to 82, andthe C-terminal capping module comprises an amino acid sequence that hasat least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or 100% sequence identity to an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 83 to 92, 111 and 112. In someembodiments, the N-terminal capping module comprises an amino acidsequence that has at least 70% sequence identity to an amino acidsequence selected from the group consisting of SEQ ID NOs: 7 to 16, theinternal ankyrin repeat(s) comprise an amino acid sequence that has atleast 70% sequence identity to an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 77 to 82, and the C-terminal cappingmodule comprises an amino acid sequence that has at least 70% sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 83 to 92, 111 and 112. In some embodiments, the N-terminalcapping module comprises an amino acid sequence that has at least 75%sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 7 to 16, the internal ankyrin repeat(s)comprise an amino acid sequence that has at least 75% sequence identityto an amino acid sequence selected from the group consisting of SEQ IDNOs: 77 to 82, and the C-terminal capping module comprises an amino acidsequence that has at least 75% sequence identity to an amino acidsequence selected from the group consisting of SEQ ID NOs: 83 to 92, 111and 112. In some embodiments, the N-terminal capping module comprises anamino acid sequence that has at least 80% sequence identity to an aminoacid sequence selected from the group consisting of SEQ ID NOs: 7 to 16,the internal ankyrin repeat(s) comprise an amino acid sequence that hasat least 80% sequence identity to an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 77 to 82, and the C-terminal cappingmodule comprises an amino acid sequence that has at least 80% sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 83 to 92, 111 and 112. In some embodiments, the N-terminalcapping module comprises an amino acid sequence that has at least 85%sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 7 to 16, the internal ankyrin repeat(s)comprise an amino acid sequence that has at least 85% sequence identityto an amino acid sequence selected from the group consisting of SEQ IDNOs: 77 to 82, and the C-terminal capping module comprises an amino acidsequence that has at least 85% sequence identity to an amino acidsequence selected from the group consisting of SEQ ID NOs: 83 to 92, 111and 112. In some embodiments, the N-terminal capping module comprises anamino acid sequence that has at least 90% sequence identity to an aminoacid sequence selected from the group consisting of SEQ ID NOs: 7 to 16,the internal ankyrin repeat(s) comprise an amino acid sequence that hasat least 90% sequence identity to an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 77 to 82, and the C-terminal cappingmodule comprises an amino acid sequence that has at least 90% sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 83 to 92, 111 and 112. In some embodiments, the N-terminalcapping module comprises an amino acid sequence that has at least 95%sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 7 to 16, the internal ankyrin repeat(s)comprise an amino acid sequence that has at least 95% sequence identityto an amino acid sequence selected from the group consisting of SEQ IDNOs: 77 to 82, and the C-terminal capping module comprises an amino acidsequence that has at least 95% sequence identity to an amino acidsequence selected from the group consisting of SEQ ID NOs: 83 to 92, 111and 112. In some embodiments, the N-terminal capping module comprises anamino acid sequence that is identical to an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 7 to 16, the internal ankyrinrepeat(s) comprise an amino acid sequence that is identical to an aminoacid sequence selected from the group consisting of SEQ ID NOs: 77 to82, and the C-terminal capping module comprises an amino acid sequencethat is identical to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 83 to 92, 111 and 112.

In some embodiments, the N-terminal capping module comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs: 7 to 16or from a variant of any one of SEQ ID NOs: 7 to 16 with up to 9, up to8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1modifications outside position 15, and wherein X at position 15 of SEQID NOs: 7 to 16 or said variants thereof is an amino acid residue asdefined above for position 15 and the internal ankyrin repeat(s)comprise an amino acid sequence that has at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or 100% sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 77 to 82 or SEQ ID NOs: 78 to 82.

In some embodiments, the N-terminal capping module comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs: 7 to 16or from a variant of any one of SEQ ID NOs: 7 to 16 with up to 9, up to8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1modifications outside position 15, and wherein X at position 15 of SEQID NOs: 7 to 16 or said variants thereof is an amino acid residue asdefined above for position 15, the internal ankyrin repeat(s) comprisean amino acid sequence that has at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, or 100% sequence identityto an amino acid sequence selected from the group consisting of SEQ IDNOs: 77 to 82 or SEQ ID NOs: 78 to 82, and the C-terminal capping modulecomprises an amino acid sequence that has at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or 100% sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 83 to 92, 111 and 112.

In some embodiments, the ankyrin repeat domain comprises the N-terminalcapping module, one internal ankyrin repeat and a C-terminal cappingmodule (N1C). Such ankyrin repeat domains are shown in Example 1. Insome embodiments, the ankyrin repeat domain comprises the N-terminalcapping module, multiple internal ankyrin repeats and a C-terminalcapping module. In one embodiment, the ankyrin repeat domain comprisesthe N-terminal capping module, 2 or 3 internal ankyrin repeats and aC-terminal capping module (N2C or N3C, respectively). In one embodiment,the ankyrin repeat domain is N2C. In another embodiment, the ankyrinrepeat domain is N3C.

In some embodiments, the protein of the invention is a recombinantprotein or DARPin.

In some embodiments, the ankyrin repeat domain of the protein of theinvention specifically binds to a target. For instance, the ankyrinrepeat domain may specifically bind to a mammalian serum albumin, suchas human serum albumin.

The protein of the invention may comprise one or more further moietiesin addition to the ankyrin repeat domain comprising the N-terminalcapping module, such as a moiety binding to a target, a labeling moiety,a toxic moiety, a moiety improving the pharmacokinetics, a moietyproviding effector functions, a moiety allowing for the purification ofthe protein or a moiety providing enzymatic activity. In someembodiments, the further moiety binding to a target is another ankyrinrepeat domain, an antibody or fragment thereof or a receptor protein. Insome embodiments, the further moiety binding to a target is anotherankyrin repeat domain. In some embodiments, the labeling moiety is astable isotope, a mass tag or a fluorescent label. In some embodiments,the toxic moiety is a chemotherapeutic agent, such as an alkylatingagent, an antimetabolite, a taxane, or an anthracycline. In someembodiments, the moiety improving pharmacokinetics is a pegylation, amammalian serum albumin, an immunoglobulin, a Fc domain of animmunoglobulin or a moiety binding to mammalian serum albumin or to animmunoglobulin. In one embodiment, the protein further contains anankyrin repeat domain binding to a mammalian serum albumin. In someembodiments, the further moiety providing effector functions is a Fcdomain of an immunoglobulin. In some embodiments, the moiety allowingfor the purification of the protein is a FLAG-tag, a GST-tag, an HA-tag,a Myc-tag, a His-tag or a Strep-tag. In some embodiments, the furthermoiety providing enzymatic or fluorescence activity is, e.g.,beta-lactamase or green fluorescence protein, respectively.

The further moiety may be proteinaceous or non-proteinaceous.

In some embodiments, the further moiety in addition to the ankyrinrepeat domain comprising the N-terminal capping module is one or moreadditional ankyrin repeat domains. In some embodiments, the protein ofthe invention comprises more than one, e.g., at least two, at leastthree, at least four or at least five, ankyrin repeat domains. In someembodiments, the protein of the invention comprises more than one, e.g.,at least two, at least three, at least four or at least five, ankyrinrepeat domains each comprising the N-terminal capping module of theinvention. In some embodiments, the protein of the invention comprisesmultiple ankyrin repeat domains which specifically bind to multipletargets. In some embodiments, the protein of the invention comprises asingle ankyrin repeat domain.

The further moiety may connect covalently to the protein, for instance,via a peptide linker or via a maleimide-containing crosslinker. Suitablepeptide linkers include glycine-serine linkers and proline-threoninelinkers. In some embodiments, the peptide linkers have a length of 2 to24 amino acid residues or 2 to 16 amino acid residues. Alternatively,the further moiety may connect non-covalently to the protein, forinstance, via a multimerization moiety. In some embodiments, amultimerization moiety is an immunoglobulin heavy chain constant region,a leucine zipper or a free thiol which can form a disulfide bond withanother free thiol.

In a further aspect, the present disclosure relates to a protein libraryof the proteins of the invention. At least two of these proteins of theprotein library differ in the amino acid sequence of their ankyrinrepeat domains. In some embodiments, the protein library has at least10³, at least 10⁵, at least 10⁷, least 10⁹, least 10¹¹ or at least 10¹³proteins that differ in the amino acid sequence of the ankyrin repeatdomain.

In some embodiments, the protein library comprises proteins of theinvention having different ankyrin repeat domain structures. Forinstance, the protein library may contain a mixture of proteins withproteins having the N-terminal capping module, 2 internal ankyrinrepeats and a C-terminal capping module and proteins having theN-terminal capping module, 3 internal ankyrin repeats and a C-terminalcapping module. In some embodiments, the proteins of the protein libraryall share the same ankyrin repeat domain structure. For instance, theankyrin repeat domain of all proteins of the library comprises theN-terminal capping module, 2 internal ankyrin repeats and a C-terminalcapping module. In other embodiments, the ankyrin repeat domain of allproteins of the library comprises the N-terminal capping module, 3internal ankyrin repeats and a C-terminal capping module. In someembodiments, the proteins of the library each comprise a single ankyrinrepeat domain only.

The sequence variability in the ankyrin repeat domains of the proteinlibrary may be brought about randomly, e.g., by error-prone PCR of thenucleic acid molecules encoding the proteins, or it may be obtained byrational design followed by, e.g., direct synthesis of the nucleic acidmolecules encoding the proteins (“design approach”). In someembodiments, the variability is introduced by the design approach. Inthe design approach, variability of the amino acid sequence isintroduced in one or more than one position of the ankyrin repeatdomains, which variable positions are also referred to as “randomizedpositions”, i.e. those positions that can potentially be occupied bymore than one amino acid residue, whereas the remaining positions remainunchanged and are also referred to as “fixed positions”, i.e. thosepositions that are occupied by a specific amino acid residue. In someembodiments, the randomized positions are those positions occupied bypotential target interaction residues and/or the fixed positions arethose positions occupied by framework residues. In some embodiments, asubset of the positions occupied by potential target interactionresidues are randomized positions. In some embodiments, all positionsoccupied by potential target interaction residues are randomizedpositions.

In certain embodiments, there are corresponding fixed positions andrandomized positions in the different proteins of the protein library.Due to the intended variability in the randomized positions, the aminoacid residues in each corresponding randomized position will usuallydiffer, although there may also be identical amino acid residues incorresponding randomized positions for at least some of the proteins inthe library (though, in such cases, the proteins will not necessarilyhave identical amino acid residues in each of their correspondingrandomized positions). In some embodiments, the fixed positions and therandomized positions are the same for the ankyrin repeat domains of eachprotein of the protein library. In some embodiments wherein the ankyrinrepeat domains have multiple internal ankyrin repeats, the internalankyrin repeats of each ankyrin repeat domain have different randomizedand fixed positions. In some embodiments wherein the ankyrin repeatdomains have multiple internal ankyrin repeats, the internal ankyrinrepeats of each ankyrin repeat domain have the same randomized and fixedpositions.

The randomized positions may show different degrees of variability, i.e.they may potentially be occupied by a different set of amino acidresidues. In some embodiments, the degree of variability differs betweenrandomized positions. In some embodiments, the amino acid residue in arandomized position is any of the twenty natural amino acid residues. Insome embodiments, a randomized position may only be occupied by a subsetof the twenty natural amino acid residues. Such subsets can be thosehaving common physicochemical properties, such as sets of hydrophobic,hydrophilic, acidic, basic, aromatic, or aliphatic amino acid residues.Other subsets are those comprising all twenty natural amino acidresidues except for certain non-desired amino acid residues, such assets not comprising cysteines or prolines. In yet other embodiments, thesubsets comprise those amino acid residues that are found in thecorresponding positions of naturally occurring ankyrin repeats.

In some embodiments, the proteins of the protein library share at least70% sequence identity, at least 80% sequence identity, or at least 90%sequence identity in the amino acid sequence of their ankyrin repeatdomains.

The above protein library can serve to select those proteins of thelibrary that have a predetermined property, i.e. a certain property ofinterest that may be found in one of the proteins of the protein libraryand that can be screened for. Such predetermined property may includethe specific binding to a target, the activation or inhibition of atarget, such as an enzyme, and the blocking of an interaction betweentwo targets. In some embodiments, the predetermined property is thespecific binding to a target.

In one embodiment, the present disclosure provides a method forselecting a protein having a predetermined property, comprising thefollowing steps:

-   -   a) Providing a protein library of said proteins comprising an        ankyrin repeat domain with the N-terminal capping module;    -   b) Selecting a protein from the library having the predetermined        property.

During the selection step b), the proteins can be selected usingscreening methods commonly known to the person skilled in the art, suchas yeast display, protein fragment complementation assay, phage displayor ribosome display. In some embodiments, the proteins are selected instep b) using phage display or ribosome display.

After the selection of a protein, the protein can be further modified,mutated and/or optimized by methods commonly known in the art.

For instance, amino acid sequence variants of the protein can begenerated, e.g., by subjecting the nucleic acid encoding the selectedprotein to physical or chemical mutagens, copying said nucleic acid byerror-prone PCR, using said nucleic acid for DNA shuffling or randomchimeragenesis (Neylon C., Nucleic Acids Res., 32(4), 1448-1459, 2004).The protein library of such amino acid sequence variants may then againbe subjected to the above selection step b) in order to select thevariant(s) having the predetermined property.

The protein selected in step b) above may also be selectively mutated.For instance, one or more cysteine residues may be introduced, the thiolgroup(s) of which can then react with maleimide cross-linkers.Similarly, certain non-desirable amino acid residues may be removed, forinstance, cysteines, which are prone to oxidations. Also, amino acidresidues may be selectively mutated after analysis of the crystalstructure so that the protein structure better fits to the target.

The protein selected in step b) may also become modified with the one ormore further moieties in addition to the ankyrin repeat domain outlinedabove for the protein of the invention.

In further aspects, the present disclosure provides a nucleic acidencoding the protein of the invention, a vector comprising such nucleicacid and a cell or in vitro expression system comprising such nucleicacid or such vector.

The nucleic acid can be DNA or RNA, single stranded or double-stranded,in isolated form or part of a larger nucleic acid, e.g., of a vector ora chromosome. The nucleic acid may comprise elements that enabledelivery of the nucleic acid to a cell and/or expression of the nucleicacid in a cell. For instance, the nucleic acid encoding the protein ofthe invention can be operatively linked to expression control sequences,which have an impact on the transcription and/or translation of theprotein, such as promoters, enhancers, transcription terminators, startcodons and stop codons. Depending on the intended application and/orcontext, the expression control sequences may be selected from anyeukaryotic or prokaryotic organism. Suitable promoters may beconstitutive or inducible promoters. Examples include the CMV-, lacZ-,T7-, T5-, RSV-, SV40-, AOX1-, and GAPDH-promoter. Suitable enhancersinclude the CMV-enhancer, insulin-responsive elements, and anSV40-enhancer. Transcription terminators include the SV40-, lacZ-, andtk-polyadenylation signal.

The present disclosure also provides a library with nucleic acidmolecules encoding the protein of the invention. At least two of thenucleic acid molecules of the nucleic acid library differ in the nucleicacid sequence of their ankyrin repeat domains. In some embodiments, thenucleic acid library has at least 10³, at least 10⁵, at least 10⁷, or atleast 10⁹ nucleic acid molecules that differ in the nucleic acidsequence coding for the ankyrin repeat domain.

The cell comprising the nucleic acid or vector encoding the protein ofthe invention can be a prokaryotic or a eukaryotic cell. In someembodiments, the cell is a bacterial, yeast or mammalian cell. In someembodiments, the cell is derived from E. coli, P. pastoris, S.cerevisiae, human, hamster or mouse. In some embodiments, the cell isselected from CHO, HEK293, BHK, NS0, Sp2/0, HT-1080, PER.C6, CAP andHuH-7 cells.

In some embodiments, the in vitro expression system comprising thenucleic acid or vector encoding the protein of the invention is based ona cell-free extract from E. coli, yeast, rabbit, wheat germ, insect orhuman.

In a further aspect, the present disclosure provides a method ofpreparing a protein comprising the following steps:

-   -   a) culturing a cell comprising a nucleic acid encoding the        protein of the invention under conditions allowing expression        thereof; and    -   b) purifying the expressed protein.

In one embodiment, the present disclosure provides a method of modifyingan existing ankyrin repeat domain by replacing the N-terminal cappingmodule of the existing ankyrin repeat domain by an N-terminal cappingmodule disclosed herein. By modifying an existing ankyrin repeat domainin this way, the favorable properties related to the N-terminal cappingmodule disclosed herein may be transferred to the existing ankyrinrepeat domain.

Thus, in one embodiment, the present disclosure provides a method ofpreparing a protein comprising an ankyrin repeat domain with an improvedthermostability, such as a higher melting temperature and/or higherfraction of refolded ankyrin repeat domains after thermal denaturation,comprising the following steps:

-   -   a) selecting a protein comprising an ankyrin repeat domain;    -   b) determining the amino acid sequence of the N-terminal capping        module of said ankyrin repeat domain;    -   c) substituting the amino acid residue in the position of the        N-terminal capping module corresponding to position 15 of SEQ ID        NO: 3 by an amino acid residue disclosed for position 15 for the        protein of the invention herein; and        -   wherein the thermostability of the resulting ankyrin repeat            protein is improved in comparison to a reference ankyrin            repeat domain having the same amino acid sequence except for            the mutation(s) in position 15 of the N-terminal capping            module.

For instance, the amino acid residue corresponding to position 15 of SEQID NO: 3 may be substituted by an amino acid residue selected from thegroup consisting of I, T, A, V, L and M.

Thus, in one embodiment, the present disclosure provides a method ofpreparing a protein comprising an ankyrin repeat domain with an improvedthermostability, such as a higher melting temperature and/or higherfraction of refolded ankyrin repeat domains after thermal denaturation,comprising the following steps:

-   -   a) selecting a protein comprising an ankyrin repeat domain        having an amino acid residue which is none of I, T, A, V, L and        M at the position of the N-terminal capping module corresponding        to position 15 of SEQ ID NO: 3;    -   b) substituting the amino acid residue in said position by an        amino acid residue selected from the group consisting of I, T,        A, V, L and M.

In one embodiment, the present disclosure provides a method of preparinga protein comprising the following steps:

-   -   a) assembling by genetic means one or more gene(s) encoding the        protein of the invention, wherein one gene comprises sequence        encoding the ankyrin repeat domain that comprises the N-terminal        capping module, followed by one or more internal ankyrin repeats        and a C-terminal capping module, and    -   b) expressing the gene(s) encoding the protein of the invention.

The present disclosure also provides a pharmaceutical compositioncomprising the protein of the invention. In some embodiments, thepharmaceutical composition comprises the protein of the invention and apharmaceutically acceptable carrier.

In a further aspect, the present disclosure provides the use of theproteins of the invention in a method of treatment.

Further Embodiments

-   -   1. A protein comprising an ankyrin repeat domain with an        N-terminal capping module, wherein the amino acid residue of        said N-terminal capping module corresponding to position 15 in        SEQ ID NO: 3 is selected from the group consisting of I, T, A,        V, L, M, S, Q and R.    -   2. A protein comprising an ankyrin repeat domain with an        N-terminal capping module, wherein the amino acid residue of        said N-terminal capping module corresponding to position 15 in        SEQ ID NO: 3 is selected from the group consisting of I, T, A,        V, L, M and S.    -   3. A protein comprising an ankyrin repeat domain with an        N-terminal capping module, wherein the amino acid residue of        said N-terminal capping module corresponding to position 15 in        SEQ ID NO: 3 is selected from the group consisting of I, T, A,        V, L and M.    -   4. A protein comprising an ankyrin repeat domain with an        N-terminal capping module, wherein the amino acid residue of        said N-terminal capping module corresponding to position 15 in        SEQ ID NO: 3 is selected from the group consisting of I, T, A,        V, L and M and the amino acid residue of said N-terminal capping        module corresponding to position 22 in SEQ ID NO: 3 is selected        from the group consisting of L, I and V.    -   5. The protein according to any one of items 1 to 4, wherein        said N-terminal capping module comprises an amino acid sequence        that is substantially identical to an amino acid sequence        selected from the group consisting of SEQ ID NOs: 7 to 16.    -   6. The protein according to any one of items 1 to 4, wherein        said N-terminal capping module comprises an amino acid sequence        that has at least 70% sequence identity to an amino acid        sequence selected from the group consisting of SEQ ID NOs: 7 to        16.    -   7. The protein according to any one of items 1 to 4, wherein        said N-terminal capping module comprises an amino acid sequence        that has at least 75% sequence identity to an amino acid        sequence selected from the group consisting of SEQ ID NOs: 7 to        16.    -   8. The protein according to any one of items 1 to 4, wherein        said N-terminal capping module comprises an amino acid sequence        that has at least 80% sequence identity to an amino acid        sequence selected from the group consisting of SEQ ID NOs: 7 to        16.    -   9. The protein according to any one of items 1 to 4, wherein        said N-terminal capping module comprises an amino acid sequence        that has at least 85% sequence identity to an amino acid        sequence selected from the group consisting of SEQ ID NOs: 7 to        16.    -   10. The protein according to any one of items 1 to 4, wherein        said N-terminal capping module comprises an amino acid sequence        that has at least 90% sequence identity to an amino acid        sequence selected from the group consisting of SEQ ID NOs: 7 to        16.    -   11. The protein according to any one of items 1 to 4, wherein        said N-terminal capping module comprises an amino acid sequence        that has at least 95% sequence identity to an amino acid        sequence selected from the group consisting of SEQ ID NOs: 7 to        16.    -   12. The protein according to any one of items 1 to 4, wherein        said N-terminal capping module comprises an amino acid sequence        that is identical to an amino acid sequence selected from the        group consisting of SEQ ID NOs: 7 to 76 and 110.    -   13. A protein comprising an ankyrin repeat domain,        -   wherein said ankyrin repeat domain comprises an N-terminal            capping module having an amino acid sequence that has at            least 70% sequence identity with an amino acid sequence            selected from the group consisting of SEQ ID NOs: 7 to 16;            and        -   wherein the amino acid residue of said N-terminal capping            module corresponding to position 15 in SEQ ID NO: 3 is            selected from the group consisting of I, T, A, V, L and M.    -   14. A protein comprising an ankyrin repeat domain,        -   wherein said ankyrin repeat domain comprises an N-terminal            capping module having an amino acid sequence that has at            least 75% sequence identity with an amino acid sequence            selected from the group consisting of SEQ ID NOs: 7 to 16;            and        -   wherein the amino acid residue of said N-terminal capping            module corresponding to position 15 in SEQ ID NO: 3 is            selected from the group consisting of I, T, A, V, L and M.    -   15. A protein comprising an ankyrin repeat domain,        -   wherein said ankyrin repeat domain comprises an N-terminal            capping module having an amino acid sequence that has at            least 80% sequence identity with an amino acid sequence            selected from the group consisting of SEQ ID NOs: 7 to 16;            and        -   wherein the amino acid residue of said N-terminal capping            module corresponding to position 15 in SEQ ID NO: 3 is            selected from the group consisting of I, T, A, V, L and M.    -   16. A protein comprising an ankyrin repeat domain,        -   wherein said ankyrin repeat domain comprises an N-terminal            capping module having an amino acid sequence that has at            least 85% sequence identity with an amino acid sequence            selected from the group consisting of SEQ ID NOs: 7 to 16;            and        -   wherein the amino acid residue of said N-terminal capping            module corresponding to position 15 in SEQ ID NO: 3 is            selected from the group consisting of I, T, A, V, L and M.    -   17. A protein comprising an ankyrin repeat domain,        -   wherein said ankyrin repeat domain comprises an N-terminal            capping module having an amino acid sequence that has at            least 90% sequence identity with an amino acid sequence            selected from the group consisting of SEQ ID NOs: 7 to 16;            and        -   wherein the amino acid residue of said N-terminal capping            module corresponding to position 15 in SEQ ID NO: 3 is            selected from the group consisting of I, T, A, V, L and M.    -   18. A protein comprising an ankyrin repeat domain,        -   wherein said ankyrin repeat domain comprises an N-terminal            capping module having an amino acid sequence that has at            least 95% sequence identity with an amino acid sequence            selected from the group consisting of SEQ ID NOs: 7 to 16;            and        -   wherein the amino acid residue of said N-terminal capping            module corresponding to position 15 in SEQ ID NO: 3 is            selected from the group consisting of I, T, A, V, L and M.    -   19. A protein comprising an ankyrin repeat domain,        -   wherein said ankyrin repeat domain comprises an N-terminal            capping module that has an amino acid sequence selected from            the group consisting of SEQ ID NOs: 7 to 16 or from a            variant of any one of SEQ ID NOs: 7 to 16 with up to 9            modifications outside position 15; and        -   wherein X at position 15 in SEQ ID NOs: 7 to 16 or said            variants thereof is an amino acid residue selected from the            group consisting of I, T, A, V, L and M.    -   20. A protein comprising an ankyrin repeat domain,        -   wherein said ankyrin repeat domain comprises an N-terminal            capping module that has an amino acid sequence selected from            the group consisting of SEQ ID NOs: 7 to 16 or from a            variant of any one of SEQ ID NOs: 7 to 16 with up to 8            modifications outside position 15; and        -   wherein X at position 15 in SEQ ID NOs: 7 to 16 or said            variants thereof is an amino acid residue selected from the            group consisting of I, T, A, V, L and M.    -   21. A protein comprising an ankyrin repeat domain,        -   wherein said ankyrin repeat domain comprises an N-terminal            capping module that has an amino acid sequence selected from            the group consisting of SEQ ID NOs: 7 to 16 or from a            variant of any one of SEQ ID NOs: 7 to 16 with up to 6            modifications outside position 15; and        -   wherein X at position 15 in SEQ ID NOs: 7 to 16 or said            variants thereof is an amino acid residue selected from the            group consisting of I, T, A, V, L and M.    -   22. A protein comprising an ankyrin repeat domain,        -   wherein said ankyrin repeat domain comprises an N-terminal            capping module that has an amino acid sequence selected from            the group consisting of SEQ ID NOs: 7 to 16 or from a            variant of any one of SEQ ID NOs: 7 to 16 with up to 4            modifications outside position 15; and        -   wherein X at position 15 in SEQ ID NOs: 7 to 16 or said            variants thereof is an amino acid residue selected from the            group consisting of I, T, A, V, L and M.    -   23. A protein comprising an ankyrin repeat domain,        -   wherein said ankyrin repeat domain comprises an N-terminal            capping module that has an amino acid sequence selected from            the group consisting of SEQ ID NOs: 7 to 16 or from a            variant of any one of SEQ ID NOs: 7 to 16 with up to 2            modifications outside position 15; and        -   wherein X at position 15 in SEQ ID NOs: 7 to 16 or said            variants thereof is an amino acid residue selected from the            group consisting of I, T, A, V, L and M.    -   24. A protein comprising an ankyrin repeat domain,        -   wherein said ankyrin repeat domain comprises an N-terminal            capping module that has an amino acid sequence selected from            the group consisting of SEQ ID NOs: 7 to 16 or from a            variant of any one of SEQ ID NOs: 7 to 16 with up to 1            modification outside position 15; and        -   wherein X at position 15 in SEQ ID NOs: 7 to 16 or said            variants thereof is an amino acid residue selected from the            group consisting of I, T, A, V, L and M.    -   25. The protein according to any one of items 19 to 22, wherein        the combined number of deletions and insertions of said variant        of any one of SEQ ID NOs: 7 to 16 is not more than 3.    -   26. The protein according to any one of items 19 to 22, wherein        the combined number of deletions and insertions of said variant        of any one of SEQ ID NOs: 7 to 16 is not more than 2.    -   27. The protein according to any one of items 19 to 22, wherein        the combined number of deletions and insertions of said variant        of any one of SEQ ID NOs: 7 to 16 is not more than 1.    -   28. A protein comprising an ankyrin repeat domain,        -   wherein said ankyrin repeat domain comprises an N-terminal            capping module that has an amino acid sequence selected from            the group consisting of SEQ ID NOs: 7 to 16 or from a            variant of any one of SEQ ID NOs: 7 to 16 with up to 9            substitutions in other positions than position 15; and        -   wherein X at position 15 in SEQ ID NOs: 7 to 16 or said            variants thereof is an amino acid residue selected from the            group consisting of I, T, A, V, L and M.    -   29. A protein comprising an ankyrin repeat domain,        -   wherein said ankyrin repeat domain comprises an N-terminal            capping module that has an amino acid sequence selected from            the group consisting of SEQ ID NOs: 7 to 16 or from a            variant of any one of SEQ ID NOs: 7 to 16 with up to 8            substitutions in other positions than position 15; and        -   wherein X at position 15 in SEQ ID NOs: 7 to 16 or said            variants thereof is an amino acid residue selected from the            group consisting of I, T, A, V, L and M.    -   30. A protein comprising an ankyrin repeat domain,        -   wherein said ankyrin repeat domain comprises an N-terminal            capping module that has an amino acid sequence selected from            the group consisting of SEQ ID NOs: 7 to 16 or from a            variant of any one of SEQ ID NOs: 7 to 16 with up to 6            substitutions in other positions than position 15; and        -   wherein X at position 15 in SEQ ID NOs: 7 to 16 or said            variants thereof is an amino acid residue selected from the            group consisting of I, T, A, V, L and M.    -   31. A protein comprising an ankyrin repeat domain,        -   wherein said ankyrin repeat domain comprises an N-terminal            capping module that has an amino acid sequence selected from            the group consisting of SEQ ID NOs: 7 to 16 or from a            variant of any one of SEQ ID NOs: 7 to 16 with up to 4            substitutions in other positions than position 15; and        -   wherein X at position 15 in SEQ ID NOs: 7 to 16 or said            variants thereof is an amino acid residue selected from the            group consisting of I, T, A, V, L and M.    -   32. A protein comprising an ankyrin repeat domain,        -   wherein said ankyrin repeat domain comprises an N-terminal            capping module that has an amino acid sequence selected from            the group consisting of SEQ ID NOs: 7 to 16 or from a            variant of any one of SEQ ID NOs: 7 to 16 with up to 2            substitutions in other positions than position 15; and        -   wherein X at position 15 in SEQ ID NOs: 7 to 16 or said            variants thereof is an amino acid residue selected from the            group consisting of I, T, A, V, L and M.    -   33. A protein comprising an ankyrin repeat domain,        -   wherein said ankyrin repeat domain comprises an N-terminal            capping module that has an amino acid sequence selected from            the group consisting of SEQ ID NOs: 7 to 16 or from a            variant of any one of SEQ ID NOs: 7 to 16 with up to 1            substitution in other positions than position 15; and        -   wherein X at position 15 in SEQ ID NOs: 7 to 16 or said            variants thereof is an amino acid residue selected from the            group consisting of I, T, A, V, L and M.    -   34. A protein comprising an ankyrin repeat domain, wherein said        ankyrin repeat domain comprises an N-terminal capping module        having an amino acid sequence that has at least 70%, at least        75%, at least 80%, at least 85%, at least 90%, at least 95%, or        100% sequence identity with SEQ ID NO: 58, and        -   wherein the amino acid residue of said N-terminal capping            module corresponding to position 15 in SEQ ID NO: 3 is            selected from the group consisting of I, T, A, V, L and M.    -   35. The protein according to any one of items 1 to 34, wherein        said N-terminal capping module further has an amino acid residue        selected from the group consisting of G, A, D and S at the        position corresponding to position 3 in SEQ ID NO: 3.    -   36. The protein according to item 35, wherein said amino acid        residue of the N-terminal capping module corresponding to        position 3 in SEQ ID NO: 3 is A or S.    -   37. The protein according to any one of items 1 to 36, wherein        said N-terminal capping module further has an amino acid residue        selected from the group consisting of L, S, Q, K, R, A, H, D and        E at the position corresponding to position 11 in SEQ ID NO: 3.    -   38. The protein according to item 37, wherein said amino acid        residue of the N-terminal capping module corresponding to        position 11 in SEQ ID NO: 3 is selected from the group        consisting of K, E, Q, A and L.    -   39. The protein according to any one of items 1 to 38, wherein        said N-terminal capping module further has an amino acid residue        selected from the group consisting of D, A, N, Q, G, S, T, K, E        and R at the position corresponding to position 17 in SEQ ID NO:        3.    -   40. The protein according to item 39, wherein said amino acid        residue of the N-terminal capping module corresponding to        position 17 in SEQ ID NO: 3 is A.    -   41. The protein according to any one of items 1 to 40, wherein        said N-terminal capping module further has an amino acid residue        selected from the group consisting of N, K, R, T, S, E, Q, A, D        and H at the position corresponding to position 20 in SEQ ID NO:        3.    -   42. The protein according to item 41, wherein said amino acid        residue of the N-terminal capping module corresponding to        position 20 in SEQ ID NO: 3 is Q.    -   43. A protein comprising an ankyrin repeat domain with an        N-terminal capping module, wherein the amino acid residue of        said N-terminal capping module corresponding to position 15 in        SEQ ID NO: 3 is selected from the group consisting of I, T, A,        V, L and M, the amino acid residue of said N-terminal capping        module corresponding to position 3 in SEQ ID NO: 3 is A or S,        the amino acid residue of said N-terminal capping module        corresponding to position 11 in SEQ ID NO: 3 is selected from        the group consisting of K, E, Q, A and L, the amino acid residue        of said N-terminal capping module corresponding to position 17        in SEQ ID NO: 3 is A and the amino acid residue of said        N-terminal capping module corresponding to position 20 in SEQ ID        NO: 3 is Q.    -   44. The protein according to any one of items 1 to 43, wherein        said N-terminal capping module further has the amino acid        residues G and S or D and A N-terminal to the amino acid        sequence corresponding to SEQ ID NO: 3 at the positions        corresponding to −2 and −1 of SEQ ID NO: 3, respectively.    -   45. The protein according to any one of items 1 to 44, wherein        said N-terminal capping module is an N-terminal capping repeat.    -   46. The protein according to any one of items 1 to 45, wherein        said ankyrin repeat domain has one or more internal ankyrin        repeats, each consisting of 33 amino acid residues.    -   47. The protein according to any one of items 1 to 46, wherein        said ankyrin repeat domain has two or three internal ankyrin        repeats.    -   48. The protein according to any one of items 1 to 47, wherein        said ankyrin repeat domain comprises one or more internal        ankyrin repeats, and        -   wherein said one or more internal ankyrin repeats each have            an amino acid sequence having at least 70% sequence identity            with an amino acid sequence selected from the group            consisting of SEQ ID NOs: 77 to 82.    -   49. The protein according to any one of items 1 to 47, wherein        said ankyrin repeat domain comprises one or more internal        ankyrin repeats, and        -   wherein said one or more internal ankyrin repeats each have            an amino acid sequence that is substantially identical to an            amino acid sequence selected from the group consisting of            SEQ ID NOs: 77 to 82.    -   50. The protein according to any one of items 1 to 49, wherein        said ankyrin repeat domain comprises one or more internal        ankyrin repeats, and        -   wherein said one or more internal ankyrin repeats each            comprise an amino acid sequence selected from the group            consisting of SEQ ID NOs: 77 to 82 or from a variant of any            one of SEQ ID NOs: 77 to 82 with up to 9, up to 8, up to 7,            up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1            modifications.    -   51. The protein according to any one of items 1 to 49, wherein        the internal ankyrin repeat of the ankyrin repeat domain that is        adjacent to said N-terminal capping module comprises an amino        acid sequence selected from the group consisting of SEQ ID NOs:        77 to 82 or from a variant of any one of SEQ ID NOs: 77 to 82        with up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to        3, up to 2 or up to 1 modifications.    -   52. The protein according to any one of items 1 to 51, wherein        said ankyrin repeat domain comprises more than one internal        ankyrin repeat, and        -   wherein said internal ankyrin repeats share at least 70%            sequence identity.    -   53. The protein according to any one of items 1 to 52, wherein        said ankyrin repeat domain further comprises a C-terminal        capping module having at least 70% sequence identity with an        amino acid sequence selected from the group consisting of SEQ ID        NOs: 83 to 92, 111 and 112.    -   54. The protein according to any one of items 1 to 52, wherein        said ankyrin repeat domain further comprises a C-terminal        capping module having an amino acid sequence that is        substantially identical to an amino acid sequence selected from        the group consisting of SEQ ID NOs: 83 to 92, 111 and 112.    -   55. A protein comprising an ankyrin repeat domain, wherein said        ankyrin repeat domain comprises an N-terminal capping module,        one or more internal ankyrin repeats and a C-terminal capping        module,        -   wherein said N-terminal capping module has an amino acid            sequence that has at least 70% sequence identity with an            amino acid sequence selected from the group consisting of            SEQ ID NOs: 7 to 16,        -   wherein the amino acid residue of said N-terminal capping            module corresponding to position 15 in SEQ ID NO: 3 is            selected from the group consisting of L, M, I, T, A, V and            S, and        -   wherein said one or more internal ankyrin repeats each have            an amino acid sequence that has at least 70% sequence            identity with an amino acid sequence selected from the group            consisting of SEQ ID NOs: 77 to 82.    -   56. A protein comprising an ankyrin repeat domain, wherein said        ankyrin repeat domain comprises an N-terminal capping module,        one or more internal ankyrin repeats and a C-terminal capping        module,        -   wherein said N-terminal capping module has an amino acid            sequence that has at least 75% sequence identity with an            amino acid sequence selected from the group consisting of            SEQ ID NOs: 7 to 16,        -   wherein the amino acid residue of said N-terminal capping            module corresponding to position 15 in SEQ ID NO: 3 is            selected from the group consisting of L, M, I, T, A, V and            S, and        -   wherein said one or more internal ankyrin repeats each have            an amino acid sequence that has at least 75% sequence            identity with an amino acid sequence selected from the group            consisting of SEQ ID NOs: 77 to 82.    -   57. A protein comprising an ankyrin repeat domain, wherein said        ankyrin repeat domain comprises an N-terminal capping module,        one or more internal ankyrin repeats and a C-terminal capping        module,        -   wherein said N-terminal capping module has an amino acid            sequence that has at least 80% sequence identity with an            amino acid sequence selected from the group consisting of            SEQ ID NOs: 7 to 16,        -   wherein the amino acid residue of said N-terminal capping            module corresponding to position 15 in SEQ ID NO: 3 is            selected from the group consisting of L, M, I, T, A, V and            S, and        -   wherein said one or more internal ankyrin repeats each have            an amino acid sequence that has at least 80% sequence            identity with an amino acid sequence selected from the group            consisting of SEQ ID NOs: 77 to 82.    -   58. A protein comprising an ankyrin repeat domain, wherein said        ankyrin repeat domain comprises an N-terminal capping module,        one or more internal ankyrin repeats and a C-terminal capping        module,        -   wherein said N-terminal capping module has an amino acid            sequence that has at least 85% sequence identity with an            amino acid sequence selected from the group consisting of            SEQ ID NOs: 7 to 16,        -   wherein the amino acid residue of said N-terminal capping            module corresponding to position 15 in SEQ ID NO: 3 is            selected from the group consisting of L, M, I, T, A, V and            S, and        -   wherein said one or more internal ankyrin repeats each have            an amino acid sequence that has at least 85% sequence            identity with an amino acid sequence selected from the group            consisting of SEQ ID NOs: 77 to 82.    -   59. A protein comprising an ankyrin repeat domain, wherein said        ankyrin repeat domain comprises an N-terminal capping module,        one or more internal ankyrin repeats and a C-terminal capping        module,        -   wherein said N-terminal capping module has an amino acid            sequence that has at least 90% sequence identity with an            amino acid sequence selected from the group consisting of            SEQ ID NOs: 7 to 16,        -   wherein the amino acid residue of said N-terminal capping            module corresponding to position 15 in SEQ ID NO: 3 is            selected from the group consisting of L, M, I, T, A, V and            S, and        -   wherein said one or more internal ankyrin repeats each have            an amino acid sequence that has at least 90% sequence            identity with an amino acid sequence selected from the group            consisting of SEQ ID NOs: 77 to 82.    -   60. A protein comprising an ankyrin repeat domain, wherein said        ankyrin repeat domain comprises an N-terminal capping module,        one or more internal ankyrin repeats and a C-terminal capping        module,        -   wherein said N-terminal capping module has an amino acid            sequence that has at least 95% sequence identity with an            amino acid sequence selected from the group consisting of            SEQ ID NOs: 7 to 16,        -   wherein the amino acid residue of said N-terminal capping            module corresponding to position 15 in SEQ ID NO: 3 is            selected from the group consisting of L, M, I, T, A, V and            S, and        -   wherein said one or more internal ankyrin repeats each have            an amino acid sequence that has at least 95% sequence            identity with an amino acid sequence selected from the group            consisting of SEQ ID NOs: 77 to 82.    -   61. A protein comprising an ankyrin repeat domain, wherein said        ankyrin repeat domain comprises an N-terminal capping module,        one or more internal ankyrin repeats and a C-terminal capping        module,        -   wherein said N-terminal capping module has an amino acid            sequence selected from the group consisting of SEQ ID NOs: 7            to 16,        -   wherein the amino acid residue of said N-terminal capping            module corresponding to position 15 in SEQ ID NO: 3 is            selected from the group consisting of L, M, I, T, A, V and            S, and        -   wherein said one or more internal ankyrin repeats each have            an amino acid sequence selected from the group consisting of            SEQ ID NOs: 77 to 82.    -   62. A protein comprising an ankyrin repeat domain, wherein said        ankyrin repeat domain comprises an N-terminal capping module,        one or more internal ankyrin repeats and a C-terminal capping        module,        -   wherein said N-terminal capping module has an amino acid            sequence that has at least 70% sequence identity with an            amino acid sequence selected from the group consisting of            SEQ ID NOs: 7 to 16,        -   wherein the amino acid residue of said N-terminal capping            module corresponding to position 15 in SEQ ID NO: 3 is            selected from the group consisting of L, M, I, T, A, V and            S,        -   wherein said one or more internal ankyrin repeats each have            an amino acid sequence that has at least 70% sequence            identity with an amino acid sequence selected from the group            consisting of SEQ ID NOs: 77 to 82, and        -   wherein said C-terminal capping module has an amino acid            sequence that has at least 70% sequence identity with an            amino acid sequence selected from the group consisting of            SEQ ID NOs: 83 to 92, 111 and 112.    -   63. A protein comprising an ankyrin repeat domain, wherein said        ankyrin repeat domain comprises an N-terminal capping module,        one or more internal ankyrin repeats and a C-terminal capping        module,        -   wherein said N-terminal capping module has an amino acid            sequence that has at least 75% sequence identity with an            amino acid sequence selected from the group consisting of            SEQ ID NOs: 7 to 16,        -   wherein the amino acid residue of said N-terminal capping            module corresponding to position 15 in SEQ ID NO: 3 is            selected from the group consisting of L, M, I, T, A, V and            S,        -   wherein said one or more internal ankyrin repeats each have            an amino acid sequence that has at least 75% sequence            identity with an amino acid sequence selected from the group            consisting of SEQ ID NOs: 77 to 82, and        -   wherein said C-terminal capping module has an amino acid            sequence that has at least 75% sequence identity with an            amino acid sequence selected from the group consisting of            SEQ ID NOs: 83 to 92, 111 and 112.    -   64. A protein comprising an ankyrin repeat domain, wherein said        ankyrin repeat domain comprises an N-terminal capping module,        one or more internal ankyrin repeats and a C-terminal capping        module,        -   wherein said N-terminal capping module has an amino acid            sequence that has at least 80% sequence identity with an            amino acid sequence selected from the group consisting of            SEQ ID NOs: 7 to 16,        -   wherein the amino acid residue of said N-terminal capping            module corresponding to position 15 in SEQ ID NO: 3 is            selected from the group consisting of L, M, I, T, A, V and            S,        -   wherein said one or more internal ankyrin repeats each have            an amino acid sequence that has at least 80% sequence            identity with an amino acid sequence selected from the group            consisting of SEQ ID NOs: 77 to 82, and        -   wherein said C-terminal capping module has an amino acid            sequence that has at least 80% sequence identity with an            amino acid sequence selected from the group consisting of            SEQ ID NOs: 83 to 92, 111 and 112.    -   65. A protein comprising an ankyrin repeat domain, wherein said        ankyrin repeat domain comprises an N-terminal capping module,        one or more internal ankyrin repeats and a C-terminal capping        module,        -   wherein said N-terminal capping module has an amino acid            sequence that has at least 85% sequence identity with an            amino acid sequence selected from the group consisting of            SEQ ID NOs: 7 to 16,        -   wherein the amino acid residue of said N-terminal capping            module corresponding to position 15 in SEQ ID NO: 3 is            selected from the group consisting of L, M, I, T, A, V and            S,        -   wherein said one or more internal ankyrin repeats each have            an amino acid sequence that has at least 85% sequence            identity with an amino acid sequence selected from the group            consisting of SEQ ID NOs: 77 to 82, and        -   wherein said C-terminal capping module has an amino acid            sequence that has at least 85% sequence identity with an            amino acid sequence selected from the group consisting of            SEQ ID NOs: 83 to 92, 111 and 112.    -   66. A protein comprising an ankyrin repeat domain, wherein said        ankyrin repeat domain comprises an N-terminal capping module,        one or more internal ankyrin repeats and a C-terminal capping        module,        -   wherein said N-terminal capping module has an amino acid            sequence that has at least 90% sequence identity with an            amino acid sequence selected from the group consisting of            SEQ ID NOs: 7 to 16,        -   wherein the amino acid residue of said N-terminal capping            module corresponding to position 15 in SEQ ID NO: 3 is            selected from the group consisting of L, M, I, T, A, V and            S,        -   wherein said one or more internal ankyrin repeats each have            an amino acid sequence that has at least 90% sequence            identity with an amino acid sequence selected from the group            consisting of SEQ ID NOs: 77 to 82, and        -   wherein said C-terminal capping module has an amino acid            sequence that has at least 90% sequence identity with an            amino acid sequence selected from the group consisting of            SEQ ID NOs: 83 to 92, 111 and 112.    -   67. A protein comprising an ankyrin repeat domain, wherein said        ankyrin repeat domain comprises an N-terminal capping module,        one or more internal ankyrin repeats and a C-terminal capping        module,        -   wherein said N-terminal capping module has an amino acid            sequence that has at least 95% sequence identity with an            amino acid sequence selected from the group consisting of            SEQ ID NOs: 7 to 16,        -   wherein the amino acid residue of said N-terminal capping            module corresponding to position 15 in SEQ ID NO: 3 is            selected from the group consisting of L, M, I, T, A, V and            S,        -   wherein said one or more internal ankyrin repeats each have            an amino acid sequence that has at least 95% sequence            identity with an amino acid sequence selected from the group            consisting of SEQ ID NOs: 77 to 82, and        -   wherein said C-terminal capping module has an amino acid            sequence that has at least 95% sequence identity with an            amino acid sequence selected from the group consisting of            SEQ ID NOs: 83 to 92, 111 and 112.    -   68. A protein comprising an ankyrin repeat domain, wherein said        ankyrin repeat domain comprises an N-terminal capping module,        one or more internal ankyrin repeats and a C-terminal capping        module,        -   wherein said N-terminal capping module has an amino acid            sequence selected from the group consisting of SEQ ID NOs: 7            to 16,        -   wherein the amino acid residue of said N-terminal capping            module corresponding to position 15 in SEQ ID NO: 3 is            selected from the group consisting of L, M, I, T, A, V and            S,        -   wherein said one or more internal ankyrin repeats each have            an amino acid sequence selected from the group consisting of            SEQ ID NOs: 77 to 82, and        -   wherein said C-terminal capping module has an amino acid            sequence selected from the group consisting of SEQ ID NOs:            83 to 92, 111 and 112.    -   69. The protein according to any one of items 1 to 68, wherein        said ankyrin repeat domain has a higher melting temperature than        a reference ankyrin repeat domain having the same amino acid        sequence except for the amino acid residue of the N-terminal        capping module corresponding to position 15 in SEQ ID NO: 3,        which is selected from the group consisting of E, D, G, H, K and        N in the reference ankyrin repeat domain.    -   70. The protein according to any one of items 1 to 68, wherein        said ankyrin repeat domain has a higher melting temperature than        a reference ankyrin repeat domain having the same amino acid        sequence except for the amino acid residue of the N-terminal        capping module corresponding to position 15 in SEQ ID NO: 3,        which is D in the reference ankyrin repeat domain.    -   71. The protein according to any one of items 1 to 68, wherein        said ankyrin repeat domain has a higher melting temperature than        a reference ankyrin repeat domain having the same amino acid        sequence except for the amino acid residue of the N-terminal        capping module corresponding to position 15 in SEQ ID NO: 3,        which is E in the reference ankyrin repeat domain.    -   72. The protein according to any one of items 1 to 71, wherein        said protein is a recombinant protein.    -   73. The protein according to any one of items 1 to 72, wherein        said ankyrin repeat domain specifically binds to a target.    -   74. The protein according to item 73, wherein said target is a        virus.    -   75. The protein according to any one of items 1 to 74, wherein        said protein comprises one or more further moieties in addition        to said ankyrin repeat domain.    -   76. The protein according to item 75, wherein said protein        comprises one or more additional ankyrin repeat domains as        further moieties.    -   77. The protein according to item 76, wherein all of said        ankyrin repeat domains comprise the same N-terminal capping        module.    -   78. The protein according to item 76 or 77, wherein said protein        comprises one or more additional ankyrin repeat domains as        further moieties that are connected by a proline-threonine        linker.    -   79. The protein according to item 78, wherein the        proline-threonine linker has a length of 2 to 24 amino acid        residues.    -   80. The protein according to any one of items 1 to 79, wherein        said protein comprises at least five ankyrin repeat domains.    -   81. The protein according to any one of items 1 to 75, wherein        said protein only comprises a single ankyrin repeat domain.    -   82. A protein library comprising more than one protein according        to any one of items 1 to 81.    -   83. The protein library according to item 82, wherein said        library comprises at least 10⁷ proteins according to any one of        items 1 to 81, wherein said proteins all differ in the amino        acid sequence of their ankyrin repeat domain.    -   84. The protein library according to item 82 or 83, wherein the        one or more proteins according to any one of items 1 to 81 share        at least 70% sequence identity in their ankyrin repeat domain.    -   85. A method for selecting a protein having a predetermined        property comprising the following steps:        -   a. providing the protein library of any one of items 82 to            84;        -   b. selecting a protein having the predetermined property            from the protein library.    -   86. A method for selecting a protein having binding specificity        to a target comprising the following steps:        -   a. providing the protein library of any one of items 82 to            84;        -   b. selecting a protein having binding specificity to the            target from the protein library.    -   87. The method according to item 85 or 86, comprising:        -   c. Further modifying the amino acid sequence of said            selected protein.    -   88. A nucleic acid molecule comprising a sequence encoding the        protein according to any one of items 1 to 81.    -   89. The nucleic acid molecule according to item 88, wherein the        nucleic acid molecule is a vector or a chromosome.    -   90. A nucleic acid library comprising more than one nucleic acid        molecule according to item 88.    -   91. A cell comprising the nucleic acid molecule according to        item 88 or 89.    -   92. A method of preparing a protein comprising the following        steps:        -   a. culturing a cell comprising a nucleic acid encoding a            protein according to any one of items 1 to 81 under            conditions allowing expression of said protein; and        -   b. purifying the expressed protein.    -   93. A method of preparing a protein comprising an ankyrin repeat        domain with an improved thermostability comprising the following        steps:        -   a. selecting a protein comprising an ankyrin repeat domain,        -   b. determining the amino acid sequence of the N-terminal            capping module of said ankyrin repeat domain; and        -   c. substituting the amino acid residue of said N-terminal            capping module corresponding to position 15 of SEQ ID NO: 3            by an amino acid residue selected from the group consisting            of I, T, A, V, L and M.    -   94. A method of preparing a protein comprising an ankyrin repeat        domain with an improved thermostability comprising the following        steps:        -   a. selecting a protein comprising an ankyrin repeat domain            having an amino acid residue which is none of I, T, A, V, L            and M at the position of the N-terminal capping module            corresponding to position 15 of SEQ ID NO: 3; and        -   b. substituting the amino acid residue in said position by            an amino acid residue selected from the group consisting of            I, T, A, V, L and M.    -   95. The method of item 93 or 94 comprising the further step of        substituting the amino acid residue of said N-terminal capping        module corresponding to position 22 by an amino acid selected        from the group consisting of L, I and V.    -   96. A pharmaceutical composition comprising a protein according        to any one of items 1 to 81 and a pharmaceutically acceptable        carrier.    -   97. A protein according to any one of items 1 to 81 for use in a        method of treatment.    -   98. Use of a protein according to any one of items 1 to 81 for        the manufacture of a medicament.    -   99. A method of treatment comprising administering the protein        according to any one of items 1 to 81 to a subject.

EXAMPLES Example 1: Effect of Mutations in the N-Terminal Capping Moduleon the Thermostability of the Ankyrin Repeat Domain

Based on crystal structure data, each position of the N-terminal cappingmodule of an ankyrin repeat domain was analyzed and predictions weremade about the most suitable amino acid residues for each position. Inlight of the inherent difficulty to correctly predict the role of themutations in the N-terminal capping module, various mutations weretested by in vitro experimentation.

Materials and Methods

Protein Sequences

Amongst others, His-tagged ankyrin repeat domains P #93 to P #109corresponding to SEQ ID NOs: 93 to 109, respectively, were tested.

The DNA sequence encoding each ankyrin repeat domain was chemicallysynthesized and cloned into pQIq (Simon M. et al., Bioconjug Chem.,23(2), 279-86, 2012) expression vectors by standard techniques.

Protein Expression

The ankyrin repeat domains were expressed in E. coli BL21 or XL1-Bluecells and purified using their His-tag using standard protocols known tothe person skilled in the art. Briefly, 25 ml of stationary overnightcultures (LB, 1% glucose, 100 mg/I of ampicillin; 37° C.) were used toinoculate 1 l cultures (same medium). At an absorbance of about 1 at 600nm, the cultures were induced with 0.5 mM IPTG and incubated at 37° C.for 4 h. The cultures were centrifuged and the resulting pellets wereresuspended in 40 ml of TBS500 (50 mM Tris-HCl, 500 mM NaCl, pH 8) andsonicated. The lysate was recentrifuged, and glycerol (10% (v/v) finalconcentration) and imidazole (20 mM final concentration) were added tothe resulting supernatant. The ankyrin repeat domains were purified overa Ni-nitrilotriacetic acid column (2.5 ml column volume) according tothe manufacturer's instructions (QIAgen, Germany). Up to 200 mg ofhighly soluble ankyrin repeat domains were purified from one liter of E.coli culture with a purity >95% as estimated from SDS-15% PAGE. Suchpurified ankyrin repeat domains were used for further characterizations.

CD Measurement

The CD signal of the ankyrin repeat domains was recorded at 222 nm in aJasco J-810 instrument (Jasco, Japan) while slowly heating the ankyrinrepeat domains at a concentration of 0.01 mM in PBS pH 7.4 from 20° C.to 95° C. using a temperature ramp of 1° C. per min. This is aneffective means to follow the denaturation of ankyrin repeat domains asthey mainly consist of alpha helices that show a strong change in theirCD signal at 222 nm upon unfolding. The midpoint of the observedtransition of such a measured CD signal trace for an ankyrin repeatdomain corresponds to its Tm value. Tm values were derived as describedin V. Consalvi et al. (Protein Eng Des Sel. 13, 501-507, 2000).

Results and Discussion

The melting curves for the above-mentioned ankyrin repeat domains weredetermined. Based on the measured melting curves, the Tm values of thevarious constructs were determined as described above.

The ankyrin repeat domain P #95 corresponding to SEQ ID NO: 95 was usedas a first exemplary reference ankyrin repeat domain. P #95 comprises aD at position 27 (which corresponds to position 15 of its N-terminalcapping module). P #96 to P #107 corresponding to SEQ ID NOs: 96 to 107,respectively, only differ in the amino acid residue at this position 15of their N-terminal capping module from P #95. FIG. 1 shows, as anexample, the melting curves of P #95 and P #101. Thus, changing a singleamino acid at position 15 (from D to V) resulted in a strong increase ofthe Tm value of the ankyrin repeat protein of about 10° C. (the Tmvalues for P #95 and P #101 were estimated to be 74.5° C. and 85.1° C.,respectively). Table 2 shows the Tm values and the corresponding aminoacids at position 15 of the respective N-terminal capping modules of P#95 to P #107.

TABLE 2 Protein SEQ ID NO: Position 15 Tm value [° C.] P#95 95 D 74.5P#96 96 L 84.6 P#97 97 M 83.8 P#98 98 I 84.8 P#99 99 T 82.3 P#100 100 A82.4 P#101 101 V 85.1 P#102 102 S 79.3 P#103 103 N 75.2 P#104 104 Q 77.4P#105 105 K 77.9 P#106 106 R 78.3 P#107 107 E 79.2

Only some of the amino acid residues in position 15, in particular I, T,A, V, L and M resulted in a profound increase of the Tm value for theankyrin repeat protein, e.g. when compared to D in this position 15.

A further reference ankyrin repeat domain (P #93) that was testedcorresponds to SEQ ID NO: 93. P #93 comprises a D at position 27 (whichcorresponds to position 15 of its N-terminal capping module). Theankyrin repeat domain P #94 corresponding to SEQ ID NO: 94 is identicalto P #93, with the exception that it contains the amino acid L at thisposition 15. FIG. 2 shows the melting curves of P #93 and P #94. Thus,changing a single amino acid at position 15 (from D to L) resulted in astrong increase of the Tm value of the ankyrin repeat protein of about13° C. (the Tm values for P #93 and P #94 were estimated to be 62.1° C.and 75.2° C., respectively).

Yet another reference ankyrin repeat domain (P #108) that was testedcorresponds to SEQ ID NO: 108. P #108 comprises a D at position 27(which corresponds to position 15 of its N-terminal capping module). Theankyrin repeat domain P #109 corresponding to SEQ ID NO: 109 isidentical to P #108, with the exception that it contains the amino acidL at this position 15. FIG. 3 shows the melting curves of P #108 and P#109. Thus, changing a single amino acid at position 15 (from D to L)resulted in a strong increase of the Tm value of the ankyrin repeatprotein of about 14° C. (the Tm values for P #108 and P #109 wereestimated to be 68.6° C. and 82.8° C., respectively).

Even though all three reference ankyrin repeat domains used (i.e. P #93,P #95 and P #109) significantly differ in their amino acid sequences oftheir N-terminal capping module (up to 9 amino acid differences outsideposition 15) a single mutation at position 15 can result in asurprisingly strong Tm increase of over 10° C. of the respective ankyrinrepeat domain.

Overall, these data show that mutations at position 15 of a N-terminalcapping module, in particular I, T, A, V, L and M, are beneficial forthe thermostability of ankyrin repeat domains, incl. designed ankyrinrepeat proteins or DARPins.

The stabilizing effect of such mutations in position 15 of theN-terminal capping module were further confirmed using different ankyrinrepeat domains.

Example 2: Effect of Mutations in the N-Terminal Capping Module on theThermostability of the Ankyrin Repeat Domain

To confirm the stabilizing effect of the mutations in the N-terminalcapping module, the mutations were tested in the context of furtherankyrin repeat proteins.

Materials and Methods

Protein Sequences and Expression

His-tagged ankyrin repeat domains P #113 to P #125 corresponding to SEQID NOs: 113 to 125, respectively, were tested.

Cloning and expression of the ankyrin repeat domains was done asdescribed in Example 1.

CD Measurement

The CD measurements of the ankyrin repeat domains P #122 to P #125 weredone as described in Example 1.

The CD signal of the ankyrin repeat domains P #113 to P #121 wasrecorded at 222 nm in a Chirascan V100 instrument (Applied Photophysics)while slowly heating the ankyrin repeat domains at a concentration of0.01 mM in PBS, 2M GdmCl, pH 7.4 from 25° C. to 100° C. using atemperature ramp of 1° C. per min, collecting data periodically at 0.5°C. intervals. Tm values were derived from the CD signal as described inExample 1.

Results and Discussion

The mutations at position 15 were transferred to three ankyrin repeatdomains with different binding specificities and largely divergingsequences. In particular, the ankyrin repeat domain P #113 correspondingto SEQ ID NO: 113 specifically binds to human serum albumin (HSA), theankyrin repeat domain P #116 corresponding to SEQ ID NO: 116specifically binds to human vascular endothelial growth factor (VEGF)and the ankyrin repeat domain P #119 corresponding to SEQ ID NO: 119specifically binds to human epidermal growth factor receptor 2 (HER2).In each of these ankyrin repeat domains, the D at position 27 of therespective sequences (which corresponds to position 15 in the N-terminalcapping module) was replaced by V and L, respectively.

The Tm values of these ankyrin repeat domains are summarized in Table 3:

TABLE 3 Protein SEQ ID NO: Target Position 15 Tm value [° C.] P#113 113HSA D 76.0 P#114 114 HSA L 84.1 P#115 115 HSA V 81.9 P#116 116 VEGF Dunfolded at RT of about 22° C. P#117 117 VEGF L 45.1 P#118 118 VEGF V 42P#119 119 HER2 D 39.6 P#120 120 HER2 L 55.7 P#121 121 HER2 V 52.6

As reflected by the above table, replacing the amino acid residue atposition 15 of the N-terminal capping module by either of V or Lincreases the thermostability of the ankyrin repeat domain in all testedbackgrounds significantly.

The particular suitability of the mutations at position 15 of theN-terminal capping module was further confirmed in different ankyrinrepeat domain backgrounds, including ankyrin repeat domain P #122corresponding to SEQ ID NO: 122 (Tm of 83.7° C.), ankyrin repeat domainP #123 corresponding to SEQ ID NO: 123 (Tm of 83.9° C.), ankyrin repeatdomain P #124 corresponding to SEQ ID NO: 124 (Tm of 81.7° C.) andankyrin repeat domain P #125 corresponding to SEQ ID NO: 125 (Tm of84.2° C.).

1. (canceled)
 2. The protein according to claim 21, wherein saidN-terminal capping module comprises an amino acid sequence that has atleast 90% sequence identity to the amino acid sequence of SEQ ID NO: 58.3. The protein according to claim 21, wherein said N-terminal cappingmodule comprises an amino acid sequence that has at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO:
 58. 4. The proteinaccording to claim 21, wherein the amino acid residue at the position ofsaid N-terminal capping module corresponding to position 15 is L.
 5. Theprotein according to claim 21, wherein said N-terminal capping modulecomprises an amino acid sequence that has at least 95% sequence identityto the amino acid sequence of SEQ ID NO: 58; and wherein the amino acidresidue at the position of said N-terminal capping module correspondingto position 15 is L.
 6. The protein according to claim 21, wherein saidN-terminal capping module further has a dipeptide GS N-terminal to thesequence having the at least 80% sequence identity with the amino acidsequence of SEQ ID NO:
 58. 7. (canceled)
 8. The protein according toclaim 21, wherein said ankyrin repeat domain comprises one or moreinternal ankyrin repeats, and wherein said one or more internal ankyrinrepeats each have an amino acid sequence having at least 80% sequenceidentity with the amino acid sequence of SEQ ID NO:
 81. 9. The proteinaccording to claim 21, wherein said ankyrin repeat domain comprises oneor more internal ankyrin repeats, and wherein said one or more internalankyrin repeats each have an amino acid sequence having at least 95%sequence identity with the amino acid sequence of SEQ ID NO:
 81. 10. Theprotein according to claim 21, wherein said ankyrin repeat domainspecifically binds to a virus.
 11. The protein according to claim 21,wherein said protein comprises one or more additional ankyrin repeatdomains as further moieties that are connected by a proline-threoninelinker.
 12. The protein according to claim 21, wherein said proteincomprises one or more additional ankyrin repeat domains as furthermoieties that are connected by a proline-threonine linker with a lengthof 2 to 24 amino acid residues.
 13. The protein according to claim 21,wherein said protein comprises at least five ankyrin repeat domains. 14.The protein according to claim 21, wherein said ankyrin repeat domaincomprises an N-terminal capping module having an amino acid sequencethat has at least 95% sequence identity with the amino acid sequence ofSEQ ID NO: 58, wherein the amino acid residue at the position of saidN-terminal capping module corresponding to position 15 is L; whereinsaid N-terminal capping module further has a dipeptide GS N-terminal tothe sequence having the at least 95% sequence identity with the aminoacid sequence of SEQ ID NO: 58; and wherein said ankyrin repeat domaincomprises one or more internal ankyrin repeats that each have an aminoacid sequence having at least 95% sequence identity with the amino acidsequence of SEQ ID NO:
 81. 15. The protein according to claim 14,wherein said protein comprises one or more additional ankyrin repeatdomains as further moieties that are connected by a proline-threoninelinker with a length of 2 to 24 amino acid residues; and wherein saidprotein comprises at least five ankyrin repeat domains.
 16. The proteinaccording to claim 14, wherein said ankyrin repeat domain specificallybinds to a virus.
 17. (canceled)
 18. (canceled)
 19. (canceled)
 20. Amethod of preparing a protein comprising an ankyrin repeat domain withan improved thermostability, the method comprising the following steps(A) or (B): (A) (i) providing a library with more than one protein, eachprotein comprising an ankyrin repeat domain that comprises an N-terminalcapping module, one or more internal ankyrin repeat(s) and a C-terminalcapping module, and (ii) selecting a protein having binding specificityto a target from the library of proteins of step (i), wherein theN-terminal capping module of the ankyrin repeat domain of said selectedprotein has an amino acid residue selected from the group consisting ofI, T, A, V, L and M at the position corresponding to position 15 andcomprises an amino acid sequence that has at least 80% sequence identitywith the amino acid sequence of SEQ ID NO: 58, and wherein thethermostability of the ankyrin repeat domain of said selected protein isimproved in comparison to a reference ankyrin repeat domain having thesame amino acid sequence except for the amino acid residue at theposition of the N-terminal capping module corresponding to position 15,which is D in the reference ankyrin repeat domain; or alternatively, (B)(i) selecting a protein comprising an ankyrin repeat domain thatcomprises an N-terminal capping module, one or more internal ankyrinrepeat(s) and a C-terminal capping module, and (ii) replacing theN-terminal capping module of said ankyrin repeat domain by an N-terminalcapping module that has an amino acid residue selected from the groupconsisting of I, T, A, V, L and M at the position corresponding toposition 15 and comprises an amino acid sequence that has at least 80%sequence identity with the amino acid sequence of SEQ ID NO: 58, whereinthe thermostability of said ankyrin repeat domain with the replacedN-terminal capping module is improved in comparison to a referenceankyrin repeat domain having the same amino acid sequence except for theamino acid residue at the position of the N-terminal capping modulecorresponding to position 15, which is D in the reference ankyrin repeatdomain.
 21. A protein comprising an ankyrin repeat domain obtained bythe method according to claim
 20. 22. The method according to claim 20,wherein the N-terminal capping module of the ankyrin repeat domain ofsaid selected protein according to the method of claim 20 (A) or theN-terminal capping module of said ankyrin repeat domain with thereplaced N-terminal capping module according to the method of claim 20(B) comprises an amino acid sequence has at least 90% sequence identitywith the amino acid sequence of SEQ ID NO:
 58. 23. The method accordingto claim 20, wherein the N-terminal capping module of the ankyrin repeatdomain of said selected protein according to the method of claim 20 (A)or the N-terminal capping module of said ankyrin repeat domain with thereplaced N-terminal capping module according to the method of claim 20(B) comprises an amino acid sequence has at least 95% sequence identitywith the amino acid sequence of SEQ ID NO:
 58. 24. The method accordingto claim 23, wherein the N-terminal capping module of the ankyrin repeatdomain of said selected protein according to the method of claim 20 (A)or the N-terminal capping module of said ankyrin repeat domain with thereplaced N-terminal capping module according to the method of claim 20(B) has L at the position corresponding to position 15 and a dipeptideGS to N-terminal to the sequence having the at least 95% sequenceidentity with the amino acid sequence of SEQ ID NO: 58; and wherein saidankyrin repeat domain of said selected protein according to the methodof claim 20 (A) or said ankyrin repeat domain with the replacedN-terminal capping module according to the method of claim 20 (B)comprises one or more internal ankyrin repeats that each have an aminoacid sequence having at least 95% sequence identity with the amino acidsequence of SEQ ID NO:
 81. 25. The method according to claim 20, themethod comprising the step (A).
 26. The method according to claim 20,the method comprising the step (B).